Preparation of Plant Material for Estimating a Wide Range of Elements

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RESEARCH NOTE No. 29 

PREPARATION OF PLANT 

MATERIAL FOR ESTIMATING 

A WIDE RANGE OF ELEMENTS 

AUTHOR 

Marcia J. Lambert 

Research Officer 

Chemistry Group 

Forestry Commission of New South Wales 

Sydney 

1976 

83311D-l

Published 1976 

Reprinted 1980 

National Library of Australia 

/ISBN 072401041 6 

ISSN 0085·3984

INDEX 

PAGE 

SUMMARY 

INTRODUCTION 

I. OXIDATION OF ORGANIC MATTER 

Dry Oxidation .. 

Wet OXidation .. 

Ashing Apparatus 

Storage of Ash Solutions 

Detailed Studies on Various Elements 

Detailed Individual Element Studies 

1. Magnesium, calcium, strontium, barium 

2. Sodium, potassium 

3. Phosphorus 

4. Aluminium 

5. Zinc 

6. Manganese 

7. Iron 

8. Copper 

Conclusions 

H. COMPARISON OF DRY ASHING PROCEDURES 

Methods and Materials 

Ashing Frocedures 

Chemical Analyses 

Results and Discussion 

Phosphorus 

Calcium, potassium 

Aluminium, magnesium, sodium 

Manganese 

Iron 

Zinc 

Conclusions 

5 

5 

5 

5 

6 

7 

7 

8 

9 

9 

9 

10 

11 

11 

12 

12 

13 

14 

15 

15 

16 

17 

17 

18 

19 

19 

19 

19 

20 

20 

HI. CRITICAL APPRAISAL OF THE SIMPLE ASHING PROCEDURE-METHOD A 21 

Methods and Materials 21 

1. Effect of Ashing Temperature 21 

2. Ash-dissolving Solutions 21 

3. Recovery Experiments 22 

Results and Discussion 23 

1. Effect of Ashing Temperature 23 

2. Ash-dissolving Solutions r;. 23 

3. Recovery Experiments 23 

CONCLUSIONS 

LITERATURE CITED 

TABLES 

APPENDICES 

25 

26 

28 

60 

3

SUMMARY 

This report is concerned with the oxidation of plant material prior 

to chemical analysis for the elements-phosphorus, aluminium, calciu~, 

magnesium, sodium, potassium, iron, zinc and manganese. The procedures 

used for the oxidation of plant material have been summarized. 

These fall into two categories-namely dry or wet ashing techniques. In 

order to obtain one solution which can be analysed for all nine elements, 

dry ashing has been shown to be preferable to wet ashing. Three dry 

ashing procedures have been fully investigated. A dry ashing procedure 

which involves removal of the insoluble siliceous residue by filtration, 

has been shown to give large errors for some elements. A commonly 

used dry ashing procedure in which the silica in the sample is destroyed, 

has also been investigated. It is a lengthy, tedious procedure and the 

results have shown that, since no greater precision is obtained, the 

removal of silica is unnecessary. A simple dry ashing procedure carried 

out at 420 0 

C with a single solution of the ash in dilute hydrochloric 

acid is the most suitable procedure for the oxidation of plant material. 

INTRODUCTION 

A research study into tree nutrition with particular reference to 

Pinus spp. was commenced by the Chemistry Group, Forestry Commission 

of N.S.W. in 1961. Dry ashing was adopted as the method for the 

destruction of organic matter prior to chemical analysis for the following 

~lements: phosphorus, aluminium, calcium, magnesium, sodium, potassium, 

iron, manganese and zinc. This dry ashing pro'cedure is a very 

tedious and lengthy process involving the separation and removal of 

silica from the ash. A more simple ashing procedure with comparable 

accuracy and precision was therefore investigated. 

This report is divided into three sections. The first part consists 

of a comprehensive literature survey covering various facets of dry ashing 

together with a discussion of the attributes of dry and wet ashing 

procedures. The second part of the report is concerned with the study 

of two simpler dry ashing procedures and their comparison with the 

dry ashing procedure which had been previously used. The third part 

is a critical appraisal of the simple dry ashing procedure which was 

found to be the most satisfactory. 

I. OXIDATION OF ORGANIC MAlTER 

There is a vast literature on the subject of the destruction of 

organic matter; this mainly falls into two categories-wet and dry oxidation. 

Both methods have their devotees and this survey covers the 

theory of the two procedures and then deals with the elements separately 

or in related groups. 

Dry Oxidation involves those procedures in which organic matter 

is oxidized by reaction with gaseous oxygen, with the application of 

energy in some form. The following series of processes is involved: 

(l) evaporation of moisture; 

(2) evaporation of volatile materials including those produced 

by thermal cracking or partial oxidation; 

5

(3) progressive oxidation of the non-volatile residue until all 

organic matter is destroyed. 

The first two steps are usually carried out at a temperature much 

lower than that used to complete the oxidation. This is largely to 

prevent·ignition of the volatile and flammable material and the subsequent 

uncontrolled rise in temperature and increased chance of loss of 

material. During the oxidation process the element to be determined 

will behave in one or more of a number of ways. Ideally it will remain 

quantitatively in the residue after oxidation and in a form in which it 

can be readily recovered, generally by solution of the ash in dilute acid. 

Some part of the element may occur in, or be converted to, a volatile 

form which may then escape from the vessel used to contain the organic 

sample. Some part of the element may combine with the vessel used 

to contain the sample, or with some other solid material in such a way 

as to be irrecoverable by the normal procedures used for the solution 

of the ash. 

Wet Oxidation procedures involve the use of some combination 

of four reagents (sulphuric acid, nitric acid, perchloric acid and hydrogen 

peroxide). Problems can arise in wet digestion procedures from 

two main sources, ,adsorption on undissolved solid material, and volatilization. 

The temperatures involved in wet oxidation methods are 

generally very much lower than in dry ashing and retention losses 

caused by reaction between the desired elements and the apparatus 

are very much less likely. The same observation applies also to reaction 

with other solid constituents of the sample. One mechanism that is 

important is coprecipitation of the element being determined with a 

precipitate fon:ned in the digestion mixture. The possibility of coprecipitation 

should be investigated. Problems can arise from reagent 

contaminat.ion and interference by acids present in solutions for subsequent 

atomic absorption analysis. Wet ashing usually requires considerable 

supervision. 

Sulphuric acid is the most frequently used component of wet 

digestion mixtures, and is widely employed in conjunction with nitric 

acid, perchloric acid and hydrogen peroxide. In addition to its function 

in partially degrading organic materials by its own action, the presence 

of sulphuric acid in mixtures containing other oxidizing agents also 

serves to raise the boiling point of the mixture and so enhance the 

action of the other ox.idants. The main disadvantages with the use of 

sulphuric acid are its tendency to form insoluble compounds and, 

ironically, its high boiling point. The high boiling point of sulphuric 

acid makes it difficult 'to remove the excess acid after completion of the 

oxidation and this is sometimes important in subsequent estimations. 

Nitric acid is much the most widely used primary oxidant for the destruction 

of organic matter. The normal concentrated acid boils at about 

120 0 C, a factor which assists in its removal after oxidation, but which 

correspondingly limits its effectiveness. Nitric acid is commonly used in 

the presence of sulphuric acid, which partially degrades the more 

resistant material, or with perchloric acid, which continues the oxidation 

after nitric acid has been 'removed. Perchloric acid has been used 

for hundreds of thousands of oxidations without incident, however the 

occurrence of occasional explosions of quite stunning violence, has led 

to it being viewed with grave suspicion. A large proportion of the 

6

explosions arose from iVhe absorption of perchloric acid by some 

materials used in the construction of fume hoods. With this awareness, 

part of the risk has been removed. There is no doubt that, handled 

with knowledge and care, this reagent is extremely efficient in the de.struction 

of organic material. From a 1echnical mther than a safety 

viewpoint, a mixture of nitric and perchloric acids probably has fewer 

disadvantages than other common wet oxidation reagents, but for use 

in unskilled or inexperienced"'hands avoidance is perhaps better than 

regret. Hydrogen peroxide is also used, particularly dn combination with 

sulphuric 'acid, and ,it is a powerful oxidizing agent. 

Ashing Apparatus. The most commonly used material for the 

construction of la:boratory apparatus is horosilicate glass, while silica, 

platinum and porcelain are also widely used for specific applications. 

Contamination can be introduced either through external contamination 

of the apparatus itself or through solution of the materiai of construction 

during the various manipulative processes. External contamination 

is generally removable by efficient acid cleaning before use. 

With regard to contamination from solution of the apparatus itself, the 

two factors of grea1est importance are the composition of the material 

and its IJ."esistance to attack by the reagents employed. The purest 

material for general use in the destruction of organdc matter is probably 

silica. In addition to its high purity, fused silica (vitreosil) is also a 

chemically resistant material, particularly at comparatively low temperatures. 

Storage of Ash Solutions. There has been considerable discussion 

of the relative merits of glass and polyethylene for the storage of dilu1e 

solutions, and Theirs 41 has concluded that unplasticized, high-pressure 

polyethylene is the best material of all. It has .the advantage of freedom 

from contaminatdon-although !the same cannot be said of the now 

common low-pressure material-and it does not appear to be unduly 

subject to adsorption problems. However, it has been shown tha:t loss 

of water vapour can occur f['Om polyethylene ampoules, although these 

losses are unlikely to be serious when considering the accuracy of most 

trace element determinations. An interesting solution to the combined 

problems of glass and polyethylene has been the use of polythene bags 

as liners for glass bottles. In general, it is probably. fair to say that 

ej,ther glass or .polyethylene can be used for these dilute solutions provided 

,that care is taken to 'achieve and maintain suitable conditions. ls 

Another important source of possible error is the loss of material 

on precipitates or other solid material produced or existing in :the body 

of the solution. At the very lowest concentration levels it is possible 

that adsorption of ions on to particles of dust or of solid substances 

in colloidal solution might cause losses, but at the part per million 

level this is not likely to be serious. More serious is the loss by adsorption 

on precipitates formed in the solution, either intentionally or 

accidently. The readiness with which precipitates which are formed in 

solutions of high pH, will carry down ions present in low concentration 

is well known, and this property is used in numerous purification and 

concentration processes. In general it is wise to avoid the formation 

of such precipitates in solutions containing only traces of ions, and 

indeed the formation of any precipitate must be viewed with suspicion. 

7

Detailed Studies on Various Elements. Gorsuch 12 conducted an 

investigation into sample loss caused by volatilization and retention of 

various elements during wet and dry oxidation. Although he found 

evidence of both volatilization and retention of various trace elements, 

he was unable to come to a definite conclusion as to whether wet or dry 

oxidation of organic matter would give the most satisfactory results 

in a particular circumstance. In a later ~omprehensive publication15 he 

concluded that for the bulk of determinations, the relative advantages 

and disadvantages of wet or dry oxidation can be weighed against 

each other on the basis of convenience, but for some applications there 

are overriding reasons for using one or the other. For example, when 

volatilization will occur during dry ashing, as with mercury and 

selenium, then wet oxidation is almost essential. When very small 

traces are to be determined in large samples the same can be said of 

dry ashing. A very pertinent statement was made in his preface-"element 

losses which can occur during wet and dry oxidation are probably 

the single greatest source of error in the majority of trace element 

determinations." 

Middleton and Stuckey29 presented a critical review of methods 

that have been used for the destruction of organic matter of different 

biological origins preparatory to the determination of trace metals. They 

pointed out that the reactions involved in dry or wet ashing vary not 

only according to the reagents and methods used, but with the nature 

of the substances concerned, and that these must be taken into account. 

It was not surprising to find that methods for destroying organic matter 

that worked satisfactorily when applied to substances of one type 

became troublesome when used for materials having quite a different 

composition: fOF example, the destruction of organic matter of vegetable 

origin and consisting largely of carbohydrates (i.e., nearly all 

vegetable tissues except seeds) by methods of dry ashing or wet oxidation 

generally offered no special problems or particular difficulty; on the 

other hand, animal tissues (organs, meat products, blood, shell-fish 

and the like) often gave trouble. 

In spite of the apparent theoretical advantages inherent in wet 

ashing, many workers have not used this procedure. Jones 24 reported 

on a survey of analytical methods for plant leaf tissue and found that 

seventeen out of the eighteen laboratories involved used dry ashing 

techniques with the majority reporting ashing times of 3 to 16 hours 

at 400 0 C to 500 0 C. 

A very recent publication 21 reported a collaborative study of wet 

and dry ashing techniques for the analysis of plant tissue by atomic 

absorption spectrophotometry. The elements determined were calcium, 

copper, iron, magnesium, manganese, potassium and zinc. The wet 

ashing was carried out with perchloric acid as the oxidizing agent. 

The dry ashing procedure involved ashing in a porcelain crucible for 

two hours at 500 0 C, digestion of the ash with dilute nitric acid and 

then solution in dilute hydrochloric acid. Both the dry and wet ashing 

techniques produced satisfactory results and the authors recommended 

either method for adoption as an official first action method. 

In a comparison between dry ashing and wet digestion in the 

preparation of plant material for atomic absorption analysis, Giron 11 

8

suggested that dry ashing is a better procedure for plant materials. This 

decision was based on the better precision for the dry ashing results 

combined with the non-significant differences between the averages of 

the two procedures. 

Detailed Individual Element Studies on a large number of elements 

have been reported and the results for those elements of importance 

to forestry are summarized below. Reference to Gorsuch 15 is not 

always acknowledged. 

1. Magnesium, calcium, strontium and barium. The removal of 

organic matter from elements of this group is reasonably simple. Their 

common salts are comparatively non-volatile and stable and they have 

no marked tendency to form volatile organic compounds or complexes. 

The choice of ashing temperature is not critical, and temperatures 

between 430· and 620· C are probably quite &atisfactory. One worker 

has reported no losses of magnesium on ashing grass samples at 430 0 , 

530 0 and 620 0 C,1° The A.O.A.C. official methods of analysis3 quotes 

temperatures between 500 0 and 550 0 C. Losses of magnesium have been 

reported at very much lower temperatures and a loss of 10 per cent 

calcium at 400 0 C has been reported. 16 A collaborative study39 on the 

analysis of poultry feed showed no significant differences in the results 

obtained after ashing at 500 0 , 600 0 , and 700 0 C and all the results 

were a little higher than those obtained by the wet ashing referee 

method. Another A.O.A.C. collaborative study19 found no significant 

difference in the recoveries of calcium and magnesium from feeds after 

dry ashing at 550 0 C for 4 hours or after wet oxidation. Roach et al. 37 

obtained excellent recoveries of magnesium from animal feeds ashed at 

450 0 C. Investigations into the recovery of strontium and barium have 

been fewer, although Thiers 41 obtained good recoveries of both at the 

one part per million level after careful ashing at 450 0 to 500 0 C. 

Wet oxidation methods have been widely used for all of these 

elements and in general little trouble has been found although caution 

is required with sulphuric acid in the various oxidation mixtures. For 

magnesium there is no problem, and for calcium in· small amounts the 

solubility of the sulphate is usually sufficient, but for samples containing 

large amounts of calcium and for strontium and barium at all times, it 

is probably wise to avoid the use of sulphuric acid altogether. Recoveries 

between 96 per cent and 100 per cent were achieved for strontium 

after oxidation with mixtures containing sulphuric acid. 12 The disadvantage 

of being unable to use sulphuric acid lies in the extent to 

;which the rate of oxidation of the sample will thereby be decreased, 

because of the lower temperatures reached. This can be overcome 

by using a mixture of perchloric and nitric acids but the possible 

hazards and precautions necessary with this mixture must be taken into 

account. 

2. Sodium and potassium. Because the alkali metals occur widely 

in ionic form, it is often possible to separate them without the necessity 

of destroying the organic matrix first. At the simplest, many of their 

organic salts are water soluble and simple solution is an adequate 

pretreatment. 

9

There appear to be no studies on ashing procedures for the determination 

of sodium and potassium in plant materials. Grove et alY 

found there was a lack of information concerning the loss of these 

elements during the dry ashing of large samp.les of animal and human 

tissue. They conducted an extensive investigation into the recovery of 

sodium and potassium from a variety of animal materials and showed 

that up to 500 0 

C, the recovery was complete even after heating for 

periods of 72 hours. At 550 0 C the recovery was complete after 24 

hours heating, but after 72 hours some were low, particularly with 

potassium, while at 650 0 

C nearly all the recoveries were significantly 

low, even at 24 hours. When the ashing temperature was increased, 

silica crucibles were attacked by the sample prQbably due to the alkali 

compounds being converted to the alkali oxides in the temperature 

range 400 0 -700 0 C. Etching of the crucibles was observed at about 

700 0 C and became more noticeable as temperature and time were 

increased. Another study25 showed that different recoveries were 

obtained for potassium when ashing was carried out in silica (95 per 

cent) and porcelain (85 per cent) crucibles. Hamilton et al. 18 reported 

negligible los.ses of sodium during the dry ashing of biological materials 

at 450°C. 

Wet oxidation methods should ·be uniformly successfup5 in obtaining 

complete recovery of the alkali metals. There is one report that 

Pyrex glass used in the construction of an oxidation apparatus adsorbed 

sodium, necessitating the use of silica5 but generally no such difficulties 

have been noted. 

Coomber and Webb 9 carried out a comparison of low temperature 

radio £requency ashing with other me.thods of organic sample oxidation 

for the determination of sodium in an acrylic fibre. Lower results were 

obtained by the two wet oxidation methods than by :the dry ashing 

procedures and they were considered to be less reliable than the dry 

methods. The concentrated acids used in wet ashing were found to 

give Jower values for sodium when present in subsequent analyses by 

atomic absorption. The two dry methods gave identical results with 

the more precise results from low temperature radio frequency ashing. 

3. Phosphorus. The results. of analyses for phosphorus using both 

wet and dry methods appear >to be identical. As early as 1936, Ashton 4 

and Weissflog and Mengdehl 43 showed that dry ashing and wet combustion 

of plant material were equally satisfactory. Ashton carried out 

the dry ashings in the presence of magnesium nitrate at a temperature 

of 800 0 C. One of the objections to dry ashing had been the contention 

that the pyro- and metaphosphates resulting from the high temperatures 

required are convert.ed with difficulty to orthophosphate. Mengdehl 28 

showed however that ten minutes of boiling with dilute hydrochloric 

acid was sufficient to convert these salts to orthophosphate. Bertramson 

7 'also obtained good recover,ies by ashing at 600°-700° C in the 

presence of magnesium nitrate. Tusl 42 studied a dry ashing procedure at 

500 0 C (for animal feeding stuffs) with and without the addition of 

sodium ca'rbonate to establish whether dry ashing without fixatives was 

adequate for the satisfactory recovery of phosphorus. He concluded 

that simple dry ashing without fixative can be used for determining 

phosphorus in biological materials. 

10

----------------------------------- 

4. Aluminium. Determinations of aluminium after destruction of 

the organic matte.r with both wet and dry oxidation proced:ures, have 

been reported and the adverse comments noted have virtually all been 

applied to the dry ash~ng methods. Dry ashing 'appears to have been 

used successfully at temperatures in the region of 500° C, although 

recovery data are sparse. In one survey41 recoveries of 95 per cent 

and 98 per cent were reported for concentrations of 1 and 3 ppm after 

ashing carefully under conditions designed to prevent contamination 

of the sample. The ash was dissolved in 6N hydrochlodc acid, and no 

difficulty was experienced, although higher ashing temperatures might 

be ,troublesome due to the increasing insolubility of the alumina produced. 

Other difficulties in recovering aluminium, have been reported,83 

when silica-contain[ng samples were ashed. The ash obtained was 

evaporated to dryness with acid, and then extracted with more acid. 

The dried residues were dissolved in hydrochloric acid, and the retained 

aluminium determined. 

5. Zinc. For ilhe estimation of zinc in organic matrices, most of 

othe common oxidation procedures have been applied, but whereas wet 

digestions seem to give uniformly good recoveries, dry ashing methods 

have aroused an appreciable amount of controversy. Ashing temperatures 

up to 900° C have been reported in the literature and most zinc 

losses have been recorded at high ashing temperatures. The two types 

of loss possible ~n dry ashing, excluding purely mechanical iosses, are 

losses by volatilization, and losses by retention, and both types have 

been postulated to explain low zinc recoveries. Gorsuch 15 investigated 

zinc volatilization and found no evidence, even under an extremely 

rigorous heating program which involved 31 hours heating at temperatures 

up to 1 000° C, that there was any volatilization of zinc. 

However, there were losses of zinc by retention on the silica ashing 

vessels. This finding was explained by the formation of a stable zinc 

silicate and it has been shown that this effect is accentuated by the 

presence of sodium chloride which apparently acts by weakening the 

silica structure and facilitating the reaction with the zinc compound. 

Gorsuch concluded that the presence of large amounts of chlorine in 

any chemical form, must be regarded with caution but apart from 

this, there was no reason why dry ashing at a temperature of 500° C 

should not be quite satisfactory. 

. On the other hand Pijck, Hoste and Gillis S4 reported a zinc recovery 

of only 30 per cent after ashing for 3 hours at 90° C. Results 

published by other investigations are no less conflicting. Hamilton, 

Minski and Cleary18 observed that no loss of zinc occurred at 850° C 

but found that 45 per cent of it was adsorbed on the silica of the 

crucible. Knauer 26 observed that there was no loss at 800°' C and 

Strohal et al. 40 obtained only' a 56 per cent recovery of zinc at 800° C. 

Roach et al. S7 obtained mean recoveries of 99 per cent from dry 

ashing animal feeds at 450° C for the determination of zinc by atomic 

absorption spectrophotometry. Raaphorst et al. s6 also recently studied 

the loss of zinc during the dry ashing of biological material. After ashing 

at temperatures of up to 1 000° C, no significant loss of,,zinc by volatilization 

was observed. Also after ashing at 450° and 5pO° C, the added 

labelled zinc-65 was removed quantitatively from the crucibles with 

hydrochloric acid. 

11

Baker and Smith 6 conducted a comprehensive investigation into 

the preparation of solutions for atomic absorption analysis of trace 

elements in plant tissue. They concluded that the high concentrations of 

extraneous ions'often present in plant ash solutions interfere with the 

determination of iron, manganese, zinc, and copper by atomic absorption. 

To overcome this, they proposed a procedure involving complete 

extraction of the elements into a separate organic phase. They stated 

that although individual elements can be determined in individual 

plant materials with little error by other procedures, their proposed 

procedure was the most reliable for all four trace elements studied. In 

the course of their work, they found dry ashing at 500 0 

C resulted in 

only small decreases in zinc levels in most of the materials and the 

mean recovery was down about 5 per cent. The recovery of zinc was 

also reduced (by 5 per cent) when it was not separated from the 

extraneous inorganic salts after wet ashing. 

6. Manganese. Both wet and dry oxidation methods have been 

applied with little adverse comment. Dry ashing temperatures up to 

at least 800 0 C have been used, but tracer recovery experiments have 

shown losses of 15 per cent at 700 0 C and 20 per cent at 800 0 C;40 so 

that upper limits of 500 0 to 550 0 C seem to be advisable. Knauer 26 

reported no losses over the temperature range 460 0 to 800 0 C. Baker 

and Smith 6 found no significant loss of manganese with dry ashing at 

550 0 C, but a small loss of 3 per cent when the manganese was not 

extracted from the residual inorganic salts after wet ashing. Loss of 

manganese by reaction with silica in the sample is sometimes considered 

to be a hazard, and the use of a low maximum temperature should 

help to minimize this type of loss. 

Wet oxidations with mixtures of nitric, perchloric and sulphuric 

acids, and hydrogen peroxide have been applied successfully,t5 but 

Bradfield 8 warned that the use of sulphuric acid should be avoided 

whenever possible in sample preparation for the determination of 

manganese by atomic absorption spectrophotometry because low values 

are obtained and there is the potential danger of precipitation of 

alkaline earth sulphates and loss of manganese by adsorption. 

An interlaboratory comparative study of wet oxidation and dry 

ashing at 550 0 

C, prior to the determination of manganese in feeds, 

revealed no significant differences between the two techniques for 

samples containing from 50 to nearly 400 ppm of manganese. 19 

7. Iron. The preparation of biological materials for the estimation 

of iron is well documented because this element is widely distributed 

and usually plays an important role in biological materials. Both wet 

and dry methods have been extensively used. Wet oxidation has proved 

very successful in almost all cases, with most of the possible combinations 

of nitric, sulphuric and perchloric acids, and hydrogen peroxide 

being used. Recovery experiments by various workers, have nearly 

always given good and consistent results. Radio-chemical measurements 

of iron at the 1 ppm level, using combinations of nitric, sulphuric and 

perchloric acids 13 have shown recoveries between 97 per cent and 102 

per cent. These results are in good accord with other radio-chemical 

work which showed recoveries of 96 per cent to 106 per cent in the 

30 to 400 ppm range 32 and ordinary chemical determinations which 

gave overall recoveries of 100± 1 per cent 22 • A serious exception to 

12

this general agreement on the suitability of wet digestion methods is 

to be found in the unsatisfactory precision obtained in a collaborative 

study by members of the AO.AC. in which six feed samples were 

analysed for iron after destruction of the organic matter by wet ashing. 3 

There is considerable disagreement however about the effectiveness 

of dry ashing. Many workers have used such methods with apparent 

satisfaction, or at least without comment, and some have reported quite 

adequate recoveries. On the other hand many have found the recovery 

varied according to the nature of the sample, the nature of the ashing 

aid and the temperature used. Satisfactory recoveries have been reported 

by a number of workers after ashing samples directly, without the use 

of an ashing aid, at temperatures in the range 450 0 to 550 0 C.44, 30, 12 

Also in the collaborative work of the AO.AC. mentioned above, 

more satisfactory precision was obtained than by wet ashing. However 

there are a great many instances of difficulties occurring during or after 

the dry oxidation of samples. 

. Knauer 26 investigated various dry ashing parameters when determining 

iron in marine shrimp, and found the optimum muffiing temperature 

range was 560 0 -600 0 C. No statistical differences were found 

when the samples were heated at 550 0 

C for periods of time ranging 

from 4 to 16 hours. In their work, Baker and Smith 6 obtained excellent 

recoveries for iron salts muffled at 550 0 

C for 4 hours. However dry 

ashing of plant tissues resulted in a 10 per cent decrease in the mean 

recovery of iron. There was the same 10 per cent loss when the iron 

was not separated from the extraneous inorganic salts after wet ashing. 

There' are two possible mechanisms for the low recovery of iron 

after the dry ashing of organic materials-loss by volatilization or loss 

by reaction with some solid material. As in the case for zinc, the 

presence of sodium chloride can greatly influence the losses due to the 

reaction 6f iron with solid material present in the systelll:. This is particularly 

the case when the ashing is carried out in silica or porcelain 

dishes in the presence of chloride ions which can greatly weaken the 

silicate structure and facilitate its reaction with iron. 13 This has been 

demonstrated in a series of experiments in which iron with Fe-59 tracer 

was heated in silica crucibles with sodium chloride; up to 40 per cent 

of the iron was retained by the crucibles. This type of loss has also 

been described by Petersen 33 who considered that it became important 

at temperatures above 600 0 

C. Other work 38 has been quoted in which 

the greatest retention losses of iron occurred on the residues of samples 

of straw and hay. The retention losses were highly correlated with the 

silica content of the samples. 

8. Copper. The wet oxidation methods appear to have been 

almost uniformly successful and nearly every possible combination has 

been used, ranging from sulphuric acid and potassium sulphate to sulphuric, 

nitric and perchloric acids plus hydrogen peroxide. However, 

there is one collaborative study by members of the AO.AC., in which 

an-investigation was made of the analysis of feedstuffs for a number of 

elements, including copper, by atomic absorption spectrophotometry. 

The results for samples prepared by wet oxidation were unsatisfactory 

and the dry ashing results were even worse. 19 

13

Dry oxidation has often been .used with apparent success. However 

there are many reports which give consistently low recoveries of 

copper. When assessments have been made of the cause of these losses, 

the usual conclusion has been that it has occurred through interaction 

between the copper and the material of the crucible. The presence of 

silica in the samples themselves is also a' serious hazard, and once 

fixation has occurred, recovery of the copper is a difficult matter. The 

normal method of solution of the ash, with hydrochloric acid, is not 

generally effective in removing the element from the ashing vessel or 

from silica present. It has been claimed a mixture of acids is more 

effective,20 and using a 2~1 mixture of dilute hydrochloric and nitric 

acids, good recoveries and agreement with wet oxidation have been 

found after ashing at temperatures between 450 0 and 600 0 C. By comparison, 

recoveries after extraction with hydrochloric acid alone were 

found to be from 15 to 60 per cent lower. The contention that the 

mixture of acids is superior was not however borne out by other work 13 

using Cu-64 as a radioactive tracer, in which no differences were found 

between the recoveries with hydrochloric acid alone, and those obtained 

with the nitric and hydrochloric acid mixture. A plausible explanation 

for the retention of copper on silica crucibles is that the reduction of 

copper to the metal occurs in the presence of organic matter and it is 

in this form that reaction with the silica takes place. It appears that 

this type of interaction is made less likely by the use of low temperatures 

and by the inclusion of an ashing aid such as magnesium nitrate. 15 

When investigating ashing temperatures, Knauer 26 obtained maximum 

sample concentration for copper in marine shrimp, between 

560 0 and 620 0 C. The report of the Analytical Methods Committee 

on the determination of small amounts of copper in organic matter 

by atomic absorption speotrophotometry pointed out that dry or wet 

ashing was suitable for the destruction of organic matter. 2 If dry 

ashing was used, the reoovery of copper should be checked and the 

residue should be dissolved in dilute hydrochloric acid or aqua regia. 

Isaac and Johnson 21 reported complete ashing of plant tissue (using 

temperatures in excess of 500 0 

C) was important for copper recovery. 

Baker and Smith 6 found a drastic loss of copper (90 per cent) on 

muffling plant tissues without the addition of phosphoric acid. 

Conclusions 

For certain elements (e.g., phosphorus, calcium and magnesium) 

both wet and dry oxidation procedures are satisfactory even when car 

1"ied out under a wide range of conditions. For the complete recovery 

of such elements as copper, iron, aluminium, sodium, potassium, and 

manganese, precise conditions for both wet and dry oxidation procedures 

are necessary. Low ashing temperatures are necessary for most 

elements-in particular sodium, potassium and aluminium. In the latter 

case, problems have been experienced with h(gh silica concentrations. 

There have also been some reports of losses of zinc, iron and copper 

by retention on silica ashing vessels. Detailed studies into wet ashing 

procedures have not been undertaken to the extent they have with dry 

ashing. Despite the apparent theoretical advantages of wet ashing, very 

few workers use it for plant material. Some losses of manganese in 

particular and sodium have heen reported in the residue remaining after 

14

wet ashing. Where the analysis of a wide range of elements is required 

on a par.ticular sample, there is little doubt that dry ashing (as opposed 

to wet ashing) provides the necessary solution for analysis, with only 

a single oxidation of the sample. , 

n. COMPARISON OF DRY ASHING PROCEDURES 

During the period 1961-70, an 'ashing procedure was followed by 

this laboratory in which the sample was charred before ashing at 

420 0 C.l The ash was digested with 5N hydrochloric acid, a small quantity 

of nitric acid added, and then taken to dryness. The residue was 

heated for 1 hour, cooled, taken up in 5N hydrochloric acid and filtered. 

The insoluble residue was washed with hot dilute hydrochloric acid until 

the washings were free of dissolved salts. The filter paper was then dried, 

ignited at 600 0 C in a platinum crucible and the silica removed with 

hydrofluoric acid in the presence of a small quantity of sulphuric acid 

according to Piper. 35 The residue was dissolved in hot dilute hydrochloric 

acid and added to the first ash solution. This procedure is 

referred to later as method C. It was considered a necessary procedure 

in order to give the maximum accuracy for the various required elements, 

some of which were analysed for the conventional colorimetric 

or titrimetric methods. However, by the introduction of more sophisticated 

instrumentation, namely the atomic absorption spectrophotometer 

(in the late 1960's), and since this ashing procedure was both 

tedious and lengthy, the possibility of finding a simpler dry ashing 

procedure was investigated. 

Destruction of organic matter by dry ashing has been reported to 

lead to the formation of insoluble silicates which may be removed 

from the sample solution dUring a filtering process. Also this l"esidue 

can contain small amounts of the mineral constituents present in the 

organic matter sample and, for some of the trace elements, this fraction 

may represent a considerable proportion of the total amount present 

in the sample. The following study was undertaken to assess a procedure 

in which the residue was left in solution on the' basis that the 

silica had been rendered insoluble and had retained minimum amounts 

of elements; the accuracy of a procedure in which the ifesidue was 

filtered off; and rfue merits of a procedure in which the silica was dissolved 

in hydrofluoric acid and then eliminated by volatilization through 

heating. 


Three ashing procedures were compared. They were assessed from 

the results obtained in the subsequent estimations of the following elements 

present in the ash solutions: phosphorus, aluminium, calcium, 

magnesium, sodium, potassium, iron, zinc and manganese. The procedures 

were: 

Method A. The ash solution was made to volume without 

separation of the insoluble siliceous material. 

Method B. The insoluble siliceous material was filtered off and 

the ash solution then made to vohllne. 

Method C. The insoluble siliceous material was filtered off, 

the silica removed with hydrofluoric acid, the residue 

solubilized, 'added to the original ash solution and 

made to volume. 

15

Twelve samples of dried finely ground foliage were selected for 

this study, comprising nine samples of Pinus radiata (D. Don), two 

of P. elliottU (Engelm.) and one P. taeda (L.). The various samples 

were chosen to cover a w.ide range of individual elemental concentrations 

as well as varying interelement relationships (e.g., high sodiumlow 

potassium and vice versa, etc.). Each sample was ashed in triplicate 

by each of the three ashing procedures (A, B and C). 

Ashing Procedures 

The following steps 1-9 in the procedure were car:ried out for 

the three methods: 

(1) a squat-shaped vitreosil crucible was oven dried for 

approximately 30 minutes at 105 0 C. A glazed, translucent 

crucible with capacity 50ml was used-No. C2 in Vitreosil 

silica catalogue; 

(2) the crucible was then cooled in a desiccator and weighed; 

(3) approximately 2g plant material was weighed accurately 

into the crucible and dried in an oven at 105 0 C overnight 

(16 hours) ; 

(4) the crucible and contents were cooled in a desiccator, 

weighed and then charred slowly under a watchglass on a 

hotplate or e~traction heating unit for about 1t hours; 

(5) the crucible and contents were then placed in ·a cool 

muffle furnace « 100 0 C) and the temperature raised 

slowly (about 100 0 C per hour) to 420 0 ± 50 C. This 

ashing temperature was maintained overnight to give a total 

ashing time of 16 hours; 

(6) .in the majority of cases, the resulting ash was greyish 

white or grey. If there were large amounts of carbon still 

remaining in the ash the sample was muffled longer (2-4 

hours) at the same temperature. After muffling, the 

crucible and contents were cooled and weighed to determine 

,the weight of crude ash; 

(7) the crucible was covered with a watch glass and the ash 

was moistened with 1-2 drops of distilled water. 3ml 5N 

hydrochloric acid was pipetted under the lip of the watch 

glass with care to avoid any loss by effervescence. The 

covered crucible was then warmed on a boiling water bath 

for 30 minutes; 

(8) 

the cover was rinsed and removed, 0.2ml 15N nitric acid 

added and the solution evaporated to dryness. The crucible 

was then placed in an oven at 105 0 C for 1 hour to complete 

the dehydration. (This treatment insolubilizes the 

silica for removal by filtering and hydrolyzes complex polyphosphates 

which can sequester iron and make it unavailable) 

; 

(9) the dried salts were moistened with 2ml 5N hydrochloric 

acid, 1amI distilled water added and warmed on a boiling 

16

water bath until all salts were in solution (about 10 

minutes). 

The following steps in the procedure were carried out for the' 

individual methods: 

Method A 

(10) the solution was transferred quantitatively (using a rubber 

tipped glass rod) to a 250ml volumetric flask with distilled 

water. The solution was then made to volume with distilled 

water; 

Method B 

(10) the solution was filtered through a Whatman No. 44 filter 

paper into a 250ml volumetric flask and the insoluble 

residue (mostly silica) was transferred using a rubbertipped 

glass rod. The filter paper was well washed with 

warm 0.02N hydrochloric acid followed by distilled water; 

( 11) the filter paper was discarded and the solution made to 

volume with distilled water. 

Method C 

(10) as for method B. 

( 11) the filter paper was placed in a platinum crucible and dried 

at 105 0 C. The filter paper was ignited by placing the 

crucible in a cool muffie and bringing it to 600 0 C quickly 

(about 200 0 C per hour). The temperature was maintained 

at 600 0 

C for half an hour; 

(12) when cool, the contents of the crucible were moistened with 

1-2 drops distilled water, 2-3 drops 36N sulphuric acid 

added and approximately 2ml concentrated hydrofluoric 

acid. This solution was heated gently until white fumes of 

sulphur trioxide appeared. Care was taken that the solution 

was not taken to dryness. More hydrofluoric acid may 

be required for samples with high silica content; 

(13) when cool, 2ml 5N hydrochloric acid and 10ml distilled 

water were added and heated gently to dissolve any insoluble 

material. The contents of the crucible were added 

to the original filtrate in the 250ml volumetric flask and 

made to volume with distilled water. 

Chemical Analyses 

The methods of chemical analysis for the estimation of phosphorus, 

aluminium, calcium, magnesium, sodium, potassium, iron, zinc and 

manganese, in the ash solutions are given in Appendices 1, 2 and 3. 


The results of the chemical analyses from the solutions obtained 

by the three ashing procedures will be discussed separately for each 

element. The various element means obtained by each ashing procedure 

are given in table 1. The mean results obtained for one bf the 

ashing procedures (method A) are given in table 2-these results give 

an indication of the wide variation in individual elements between the 

foliage samples selected for this study. 

8331lD-2 

17

The :results for phosphorus obtained by the three ashing methods 

are shown !in table 3. Analysis of variance (table 3a) was used to 

investigate whether there were any statistical differences between the 

ashing methods. The results gave no statistical differences between 

methods, mainly because of overlap between all three methods. The 

above findings are in very good agreement with those of the literature 

in which dry ashing under a range of conditions was found to be suitable 

for the estimation of phosphorus. It is important to note from 

these results that there is no positive interference in the colorimetric 

estimation of phosphorus because of the presence of silica in the ash 

solution (method A). The ashing procedures involve a careful dehydration 

of the silica and ensure the silica is completely insoluble and 

hence in a form in which it does not interfere with the phosphorus 

estimation-it is only soluble. silica whioh interferes. 1t is, therefore, 

apparently not necessary to remove the silica from solution such as is 

carried out in methods Band C and followed by many workers. 

The three ashing methods also gave statistically inseparable results 

for calcium and potassium (tables 4, 4a and 5, Sa respectively). The 

findings for calcium were supported by the literature from which it 

was concluded that the oxidation of organic matter presents no problems 

pr,ior to the analysis of calcium. Similarly there are no reported 

difficulties for po'tassium provided low ashing temperatures are used. . 

The results for aluminium, magnesium, sodium and manganese 

are tabulated in tables 6, 7, 8 and 9 respectively. Analysis of variance 

was carried out for each set of data (tables 6a, 7a, 8a and 9a respectively). 

Analysis of variance using all the aluminium results (table 

6a) was complicated by a very significant interaction term. The magnitude 

of this interaction term was due largely to the results obtained 

for the samples from Murraguldrie, Mannus 'and Barcoongere which 

were inconsistent with the results obtained for the other samples. 

However, there were significant differences between the three methods 

when they were sta,tistically analysed in pairs-method C gave the 

highest results (mean = 776 ppm AI) followed by method A (mean 

= 739 ppm AI) with the lowest resu1ts being obtained by method 

B (mean =708 ppm AI). In the case of magnesium, analysis of 

variance (table 7a) gave no significant difference between method A 

and method C whereas method B was significantly lower (the mean 

difference being 5 per cent) than the other two ashing methods. There 

was also very considerable variation between the replicates in method 

B, whereas the replicate variation in methods A and C was very small. 

A very significant interaction term (due to inconsistencies within 

methods B and C) was obtained in the analysis of the results for 

sodium (table 8a) and hence the results for the individual methods 

were statistically analysed, two methods at a time. For the majority of 

samples, the highest sodium values were obtained by method A with 

method C giving the lowest results. Although sometimes the results 

obtained for a particular sample were higher by method B than method 

C or vice versa, the mean results for the two methods were the same 

(313 ppm Na and 316 ppm Na respectively). However, there was 

a mean 12 per cent loss when compared with the results from method 

A. The results of the statistical :analysis for manganese (table 9a) gave 

method C 3 per cent significantly higher than method A, with method B, 

18

8 per cent lower than the other two methods. However, the interaction 

between the methods was significant mainly because of the large 

variat~on within the results obtained by method B. 

For each of these four elements (aluminium, magnesium, sodium 

and manganese), method B gave the lowest results. The elements 

appear to fall

these results for iron. The lack of difference between methods A and B 

indicates either that there is no adsorption of iron on the silica in the 

sample or more likely that it is adsorbed and insoluble in the solution 

prepared according to method A, and ,then filtered off by method B. 

Hence the results from these two methods were the same. With the 

separation from silica in method C, higher iron results were then 

obtained. It is worth noting however, that for some samples there was 

considerable variation in the results obtained for the replicates by 

method C. Also not all samples gave differences between methodsfor 

25 per cent of the samples there was no difference in the mean 

results for methods A and C. 

The mean results obtained for zinc by the three methods were 

very similar (table 11). Methods A and B both had mean levels of 

65 ppm zinc, with method C 5 per cent lower (table lla) at 62 ppm 

zinc. However, the results within method B were extremely variable 

and had to be excluded to give the above finding without the complication 

of a significant interaction term. There is a vast amount of 

literature on the subject of the preparation of biological materials for 

the analysis of zinc and the majority of workers found no problems 

over a range of ashing temperatures. The difference found for method 

C, although small, is hard to explain but is most probably a combination 

of the extra variation obtained with the filtering step and the second 

ashing at 600° C. 

Some further results of analyses of ash solutions prepared (from 

foliage samples from Nalbaugh State Forest) according to methods A 

and C are given in table 12. The results for all the elements are in 

agreement with those discussed above. 

Conclusions 

The results of the investigation into the three dry ashing procedures 

have shown that the simple foliar ashing procedure referred to 

as method A, gave a solution of the ash which when analysed for the 

nine elements was found to give more consistent results than either the 

other simple ashing procedure method B, or the more tedious, lengthy 

procedure of method C. 

For methods A and C, the analyses for the elements phosphorus, 

calcium, magnesium and potassium gave identical results. The analyses 

for sodium and zinc were found to be 5 per cent more accurate by 

method A, whereas those for aluminium, iron and manganese were 5, 

3 and 3 per cent respectively underestimated by method A. Every 

step in an analytical procedure is a potential source of error and it 

must not be overlooked that apart from the saving in time (50 per 

cent) of the simplified ashing procedure when compared to the more 

tedious one, the simple procedure has been shown to be more consistent. 

There is minimal variation between replicates and this is in contrast to 

the lengthy silica removal procedure or the filtration procedure in 

particular which resulted in considerable variation-particularly for 

certain elements such as aluminium, sodium, iron and manganese. 

Hence, although the results from methods A and C were very 

similar over the range of the nine elements studied, the results obtained 

20

from method B were often quite markedly different. Results comparable 

with methods A and C were obtained by method B for phosphorus, 

calcium, and potassium, but sodium, iron and zinc were underestimated 

by 5, 3 and 3 per cent respectively, and there were even larger errors 

with aluminium, manganese and magnesium being 9, 9 and 5 per cent 

respectively. The worst aspect of this method was that a large variation 

was obtained between replicates. Many workers follow an ashing procedure 

in which the ash solution is filtered and the silica residue discarded 

and as shown by the preceding results this could give a solution 

for analysis which would be far from satisfactory. However by leaving 

the silica in solution (method A), minimal errors occur in that the 

elements (particularly magnesium) can solubilise on standing in the 

acid solution. In this case, the error incurred is less than the variation 

which can be expected with the lengthy silica removal procedure of 

methodC. 

It is recommended that the ashing procedure of method A be 

adopted for the destruction of organic matter in plant material. The 

next section of this report is a critical appraisal of method A. 

Ill. CRITICAL APPRAISAL OF THE SIMPLE ASHING PRO 

CEDURE-METHOD A 

The following investigations were undertaken in order to assess 

the accuracy of method A as an ashing procedure for the destruction 

of organic matter in plant material. In all cases the procedure outlined 

as method A in section II was followed. 


1. Effect of Ashing Temperature 

The simple ashing procedure was assessed over the temperature 

range 350 0 to 550 0 C to determine whether there were any element 

losses dependent on the ashing temperature. These losses were considered 

possible by volatilization or by such means as adsorption on 

the silica crucibles. A homogeneous sample of P. radiata foliage was 

prepared for this study and samples were ashed in triplicate at various 

temperatures-350° C, 400 0 C, 425 0, 450 0 C, 500 0 C, 525 0 C, 

550 0 C. 

2. Ash-dissolving Solutions 

From the literature it appeared evident that the degree of adsorption 

of certain elements on the silica crucible could be affected by the 

nature of the ash-dissolving solutions. Four different ash-dissolving 

solutions were investigated. The bulk foliage sample used for this investigation 

was the same as that above. The various samples were ashed 

following the procedure of method A, however a muffling temperature 

of 550 0 C was used rather than 420 0 C. This ashing temperature was 

chosen since the adsorption of some elements on the silica of the 

crucible appears to be enhanced at high temperatures, therefore any 

significant differences in the ash-dissolving solutions would be more 

21

evident at this temperature. In step 7 of the ashing procedure, the 

following ash-dissolving solutions were used: 

(a) 3ml 5N HCl; 

(b) 3ml 5N HN03; 

(c) 3ml dilute HCl/HN03 (prepared by mixing equal volumes 

of 5N acids); 

(d) two digestions (followed by evaporation) of the dried salts 

with 3ml 5N HC!. 

In each case, for step 9, the residue was dissolved in 2ml of the 

same acid as that used to dissolve the ash. 

3. Recovery Experiments 

The accuracy of the simple ashing procedure was investigated by 

car,rying out recovery experiments. The recover,ies of phosphorus, alu~ 

minium, calcium, magnesium, sodium, potassium, iron, zinc and man· 

ganese were assessed over a range of ashing temperatures. They were 

also assessed with and without the presence of added silica, since it 

was considered possible that the quantity of silica within a sample 

could affect the quantitative estimation of some elements. 

The ,recovery tests were carried out on 2g subsamples of the 

homogeneous sample of P. radiata foliage. A 5ml aliquot of a standard 

solution (see below) was added to particular subsamples. The 

relative levels of the various elements in the standard solution were 

chosen to correspond with those usually obtained from healthy 

P. radiata foliage. After the addition of the standards, the solutions were 

evaporated to dryness before proceeding with the ashing procedure 

(step 4). The standard solution was prepared so that a 5ml aliquot 

contained the following elemental concentrations: 

Al 

Ca 

Mg 

Mn 

Zn 

Fe 

2 000 p.g per 5m!. 

10000 p.g per 5m!. 

10 000 p.g per 5m!. 

500 p.g per 5ml. 

100 p.g per 5m!. 

500 p.g per 5m!. 

Phosphorus and potassium were added as individual weights of 

crystalline KH 2 P04 to give phosphorus additions of between 4 000 p.g 

and 5000 p.g P. 3000 p.g potassium was added to each sample and with 

the potassium contribution from the KH2P04, the potassium additions 

were in the range 8 030 p.g to 9 300 p.g. The silica additions were 

made with 19 weights of pure silica. The standard solution was prepared 

to contain 500 p.g sodium per 5m1 solution. However, this was estimated 

by atomic absorption to be actually 583 p.g sodium. This was 

expected as sodium is an impurity in many AR. inorganic reagents. 

The actual concentrations of all elements in the standard solution were 

determined accurately (see appendix 1 for analytical methods). 

22

The conditions for the various recovery experiments were 'as follows: 

No. 

I 

Sample 

Ashing temperature 

QC 

1 

2 

Standard Solution .. 

Standard Solution + Silica .. 

.. 1 Not muffled 

., Not muffleJ 

----1-------------------·------,-,----- 

3 Standard Solution . . . . . . 

420 

4 Standard Solution + Silica .. . . 

420 

5 Foliage . . . . . . .. 

420 

6 Foliage Standard Solution .. 

420 

7 Foliage + Standard Solution + Silica 

420 

8 

9 

10 

11 

12 

Standard Solution . . . . 

Standard Solution + Silica .. 

Foliage.. .. .. .. 

Foliage + Standard Solution 

Foliage + Standard Solution + Silica 

18 Standard Solution . . . . . . 

19 Standard Solution + Silica .. . . 

20 Foliage.. .. .. .. ., 

21 Foliage + Standard Solution .. 

22 Foliage + Standard Solution + Silica 

500 

500 

500 

500 

500 

550 

550 

550 

550 

550 

600 

600 

600 

600 

600 


1. Effect of Ashing Temperature 

The results obtained for the nine elements are shown in tables 

13-22. Over the ashing temperature range of 350° to 550° C, significant 

differences between temperatures were only obtained for the elements 

calcium and potassium. In the case of calcium, significantly lower 

results were obtained at 350° and 550° C, while the same results were 

obtained for all the other ashing temperatures. The losses only amounted 

to 2 per cent and this is considerably less than the 10 per cent loss at 

400.0 C reported by Griggs et al. 16 There was a significant loss of 

potassium for all ashing temperatures above and including 450 0 C. 

This is most probably by volatilization. The difference obtained by 

increasing the temperature from 425° to 450° C amounted to a loss of 

8 per cent potassium but as the ashing temperature was raised to 550 0 C 

there was no further loss. There is only one report of losses of potassium 

up to 500 0 

C where, at the trace level, the various chemical forms 

of the five alkali metals showed considerable losses up to 600° C.27 

In a study on ashing grass samples, Davidson 1o reported losses of 7 per 

cent at 740° C whereas temperatures of 430°,530° and 620° C caused 

no losses. 

For some of the other elements, although there were no statistical 

differences between ashing temperatures, there are other pertinent 

23 

L 

·_

observations. Considerable variation and lower results (almost significant) 

were obtained for aluminium at the lower temperatures of 

350°,400° and also 550° C. It has been considered 41 that higher ashing 

temperatures might be troublesome due to the decreasing solubility of 

the alumina produced. There was a tendency towards higher iron 

results with increasing temperature. This is in agreement with Knauer 26 

and although not significant, the higher results obtained at 500-550° C 

do offer a possible explanation for the higher iron results obtained by 

method C when compared with method A (in section B). 

Muller 31 investigated the effect of different ashing temperatures on 

the macroelements in pine needles and chose a maximum of 450 0 

or 

preferably 425° ± 25° C as a suitable ashing temperature. 

Since at ashing temperatures below 425° C calcium is underestimated, 

and above 450° C there are considerable losses of potassium 

which outweigh the small increases in iron above 550° C, it can be 

concluded from this investigation of ashing temperatures that the optimum 

temperatures for ashing plant material are between 425 0 

and 

450° C. 

2. Ash-dissolving Solutions 

These results are given in tables 23-32. The only element for 

which there was a significant difference between the four ash-dissolving 

solution was zinc. Every other element gave statistically inseparable 

results. There have been reports of losses of zinc by retention'on the 

silica ashing vessels. 15 This present work has shown that a single 

digestion with dilute hydr.ochloric acid is the most effective means of 

minimizing this retention. However it would appear that with repeated 

digestions with dilute hydrochlo;ic acid or digestion with dilute nitric 

acid, enhanced retention of the zinc will occur. In these cases, errors 

of between 5 and 10 per cent were obtained. It does appear that the 

lack of difference between ash-dissolving solutions for the other elements 

indicates there is minimal adsorption of these elements on to the 

silica ashing crucible. 

3. Recovery Experiments 

The results of the recovery work are given in table 33. Those for 

the foliage (only) samples have been reported as actual determinations 

for each element while those for the standard solution (elements) are 

expressed as percentage recoveries based on the added amounts. 

The results to be considered firstly are those from the foliage-only 

samples. As would be expected comparable results were obtained, over 

the studied temperature range, with the work reported earlier on ashing 

temperatures. That is, with increasing ashing temperature, losses occurred 

for calcium, potassium, aluminium and zinc. However the major 

losses were primarily at 600° C-a higher ashing temperature than 

studied earlier. 

The accuracy of method A can be assessed from the results 

obtained at the ashing temperature of 420° C. Iron is the only element 

which appears to give less than satisfactory recovery but the results 

for iron are variable over the whole of the temperature range studied. 

24

With the lack of replicates at each temperature it is difficult to draw 

definite conclusions about this element. The basic uniformity of the 

results for the other elements overcomes the lack of replicates, since 

each temperature level represents a replicate. 

Different recoveries for some elements have been obtained depending 

on the presence or absence of added silica. Considering the large 

amount of silica added (lg), the recoveries are remarkably good. 

Over the range of ashing temperature from 420° to 600° C, the elements 

most affected by added silica were calcium, sodium, iron, zinc, 

and manganese. The general trend was for losses to increase with 

increasing ashing temperature. The findings for zinc and manganese 

were in agreement with reports in the literature on the possible retention 

of these elements on silica present in the foliage sample. However 

these present recovery tests were carried out in the presence of a very 

large amount of added silica which is far in excess of that present in 

pine foliage material. It is obvious that the recoveries of the various 

elements in the presence of foliage are excellent and hence the concentration 

of silica normally present in pine foliage presents no problems. 

When samples known to contain high concentrations of silica (e.g., 3 

to 4-year-old needles from some coniferous species) are to be ashed, 

there is the possibility of some losses. 

The most important finding from this recovery work is that the 

accuracy of ashing method A is excellent and hence there is no need 

to follow an ashing procedure which involves the removal of silica for 

pine foliage. It can also be generalized that possible losses by retention 

of some elements on the silica of the ashing vessel are minimalparticularly 

in comparison to possible losses by adsorption on silica in 

high silica-containing samples. 

Conclusions 

It has been shown that the accuracy of the simpl~ ashing procedure 

referred to as method A is excellent. The optimum ashing temperature 

is 425°C and provided the ash ·is dissolved in a single digestion of 

dilute hydrochloric acid, optimum accuracy is obtained in the determination 

of the required inorganic constituents in the plant materialnamely 

phosphorus, aluminium, calcium, magnesium, sodium, potassium, 

iron, zinc and manganese. The accuracy of the procedure has 

been verified by recovery tests, from which it was also conduded that 

some losses can possibly occur during the dry ashing of plant material 

which is known to contain high silica concentrations (e.g., this may be 

possible with some coniferous species other than pine) . 

25

LITERATURE CITED 

1Analytical Methods Committee. 1960. Methods for the destruction of 

organic matter. Analyst 85:643-656. 

2 Analytical Methods Committee. 1971. The determination of small amounts 

of copper in organic matter by atomic-absorption spectroscopy. Analyst 96:741 

743. 

:3 Association of Official Analytical Chemists. 1965. Official methods of 

analysis. 10th Edn. 

4 Ashton, F. L. 1936. Influence of the temperature of ashing on the accuracy 

of the determination of phosphorus in grass. J. Soc. Chem. Ind. 55:106T-I08T. 

5Aubouin, G. 1963. Analyse par activation d'impureties minerales dans les 

terphenyles et leur dosage par spectrometrie. Radiochimica Acta 1: 117-123. 

6 Baker, A. S. and R. L. Smith. 1974. Preparation of solutions for atomic 

absorption analyses of Fe, Mn, Zn and Cu in plant tissue. J. Agr. Food Chem. 

22:103-107. 

7 Bertramson, B. R. 1942. Phosphorus analysis of plant material. Plant 

Physiology 17:447-454. 

8 Bradfield, E. G. 1942. Chemical interferences in the determination of 

manganese in plant material by atomic absorption spectroscopy. Analyst 99:403 

407. 

9 Coomber, R. and J. R. Webb. 1970. Comparison of low temperature 

radiofrequency ashing with other methods of organic sample oxidation for the 

determination of sodium in an acrylic fibre. Analyst 95:668-669. 

10 Davidson, J. 1952. The micro-determination of magnesium in plant 

materials with 8-hydroxyquinoline. Analyst 77:263-268. 

11 Giron, H. C. 1973. Comparison between dry ashing and wet digestion 

in the preparation of plant material for atomic absorption analysis. Atomic 

Absorption Newsletter 12:28-29. 

12 Gorsuch, T. T. 1959. Radiochemical investigations on the recovery for 

analysis of trace elements in organic and biological materials. Analyst 84:135-173. 

13 Gorsuch, T. T. 1960. Ph.D. Thesis, Univ. of London. 

14 Gorsuch, T. T. 1962. Losses of trace elements during oxidation of 

organic materials. Analyst 87: 112-115. 

15 Gorsuch, T. T. 1970. The destruction of organic matter. Pergamon 

Press 151 pp. 

16 Griggs, M., R. Johnstin, and B. Elledge. 1941. Mineral analysis of 

biological material. Ind. Eng. Chem. (Anal. Ed.) 13 :99-101. 

17 Grove, E. L., R. A. Jones, and W. Mathews. 1961. The loss of 80dium 

and potassium during the dry ashing of animal tissue. Anal. Biochem. 2:221-230. 

18 Hamilton, E. I., M. J. Minski, and J. Cleary. 1967. The loss of elements 

during the decomposition of biological materials with special reference to arsenic, 

sodium, strontium and zinc. Analyst 92:257-259. 

19 Heckman, M. 1967. Minerals in feeds by atomic absorption spectrophotometry. 

J. Ass. Off. Anal. Chem. 50:45-49. 

20 High, J. H. 1947. The determination of copper in food. Analyst. 72:60-62. 

21 Isaac, R. A. and W. C. Johnson. 1975. Collaborative study of wet and 

dry ashing techniques for the elemental analysis of plant tissue by atomic 

absorption spectrophotometry. J. Ass. Off. Anal. Chem. 58:436-440. 

22 Jackson, S. H. 1938. Determination of iron in biological material. Ind. 

Eng. Chem. (Anal. Ed.) 10:302-304. 

26

23 Jones, L. H. P. and A. A. Milne. 1963. Studies of silica in the oat plant. 

I. Chemical and physical properties of the silica. Plant and Soil 18:207-220. 

24 Jones, J. B. 1969. Elemental analysis of plant leaf tissue by several 

laboratories. J. Ass. Off. Anal. Chem. 52:900-903. 

25 Joyet, G. 1951. Method for determining relative dosage of biological 

samples. Nucleonics. 9:42-49. 

26 Knauer, G. A. 1970. The determination of magnesium, manganese, iron, 

copper and zinc in marine shrimp. Analyst 95 :476-480. 

27 Kometani, T. Y. 1966. Effect of temperature on volatilization of alkali 

salts during dry ashing of tetrafluoroethylene fluorocarbon resin. Anal. Chem. 

38: 1596~1598. 

28 Mengdehl, H. 1933. Studien zum phosphorstoffwechsel in der hoheren 

pflanze. I. Die Bestimmung von pyro- und metaphosphate, sowie von phosphit 

und hypophosphit in pflanzenmaterial. Planta. 19:154-169. 

29 Middleton, G., and R. E. Stuckey. 1953. The preparation of biological 

material for the determination of trace metals. Part 1. A critical review of 

existing procedures. Analyst' 78 :532-542. 

30 Morgan, L. 0., and S. E. Turner. 1951. Recovery of inorganic ash from 

petroleum oils. Anal. Chem. 23:978-979. . 

31 Muller, W. 1970. A method for determining the macro- and micronutrients 

in conifer needles. Arch. Forstwesen. 19:861-876. 

32 Nicholas, D. J. D., C. P. Lloyd-Jones, and D. J. Fisher. 1957. Some 

problems associated with determining iron in plants. Plant and Soil 8:367-377. 

33 Petersen, R. E. 1952. Separation of radioactive iron from biological 

materials. Anal. Chem. 24: 1850-1852. 

34 Pijck, J., J. Hoste, and J. GiIlis. 1958. Int. Symp. on Microchem., 

Birmingham. 

35 Piper, C. S. 1950. Soil and plant analysis. The University of Adelaide. 

36 Raal'horst, J. G. van, A. W. van Weers, and H. M. Haremaker. 1974. 

Loss of zinc and cobalt during dry ashing of biological material. Analyst 99: 

523-527. 

37 Roach, A. G., P. Sanderson and D. R. WilIiams. 1968. Determination of 

trace amounts of copper, zinc and magnesium in animal feeds by atomicabsorption 

spectrophotometry. Analyst 93:42-49. 

38 Sandell, E. B. 1944. Colorimetric determination of traces of metals. 

lnterscience, New York. 

39 St John, J. L. 1941. Effects of ashing temperatures on percentage of 

mineral elements retained. J. Ass. Off. Anal. Chem. 24:848-854. 

40 Strohal, P., S. Lulic, and O. Jelisavcic. 1969. The loss of cerium, cobalt, 

manganese, protactinium, ruthenium and zinc during dry ashing of biological 

material. Analyst 94:678-680. 

41 Thiers, R. E. 1957. Methods of biochemical analysis Vol. 5. p. 273. 

lnterscience, New York (D. Glick, editor). 

42 Tusl, J. 1972. Spectrophotometric determination of phospohrus in biological 

samples after dry ashing without fixatives. Analyst 97: 111-113. 

43 Weissflog, J., and H. Mengdehl. 1933. Studien zum phosphorstoffwechsel 

in der hoheren pflanze. III Aufnahme und verwertbarkeit organischer phosphorsaureverbindungen 

durch die pflanze. Planta 19: 182-241. 

44 Wyatt, P. F. 1953. Diethylammonium diethyldithio-carbamate for the 

separation and determination of small amounts of metals. Analyst 78:656-661. 

27

TABLE 1 

Mean foliar chemical analyses obtained for the three ashing 'procedures 

N 

00 

Element 

Method A 

ppm 

Method B 

Method C 

Significance 

Level between 

Methods 

I 

I I I I 

Significant 

difference 

required between 

means (ppm) 

Phosphorus .. .. .. .. .. .. 1551 1519 1546 

Aluminium .. .. .. .. .. .. 739 708 776 

Calcium .. .. .. .. I * 31 

.. .. .. 2161 2140 2160 

Magnesium .. .. .. .. .. .. 1657 1568 

Sodium 

1644 

.. .. .. 36 

.. .. .. .. 357 313 316 

Potassium .. .. * 10 

.. .. .. .. 6688 6611 

Iron 

6519 .. .. .. .. .. .. .. 

182 182 

Zinc 

189 

.. .. 5 

" .. .. 

" .. 65 65 

Manganese 

62 

.. .. 1 

.. .. .. .. 237 223 245 * 7 

* Significant at 1 per cent level.

TABLE 2 

Mean foliar chemical analyses obtained for the separate samples ashed according to method A 

Forest 

Species 

p Al Ca Mg 

ppm 

Na K Fe Zn Mn 

t'd 

Mullions Range ..1 P. radiata ·. 796 791 945 1075 96 6348 255 53 101 

Old Depot ·. .. P. radiata ·. 2006 556 2164 1 107 662 5483 234 79 206 

Penrose ·. .. P. radiata ·. 978 867 4033 2234 157 6002 199 85 284 

Jervis Bay .. P. radiata ·. 1801 781 1191 955 1933 8780 344 37 36 

Murraguldrie I .. P. radiata ·. 3410 733 3234 3076 29 5521 183 102 317 

Belanglo ·. .. P. radiata ·. 1063 403 1820 1684 157 6713 211 35 139 

Mannus ·. .. P. radiata .. 2581 902 2927 3107 58 5977 169 95 448 

Lidsdale ·. .. P. radiata ·. 1668 942 2351 1941 181 8919 149 84 376 

Newnes ·. .. P. radiata ·. 1212 746 796 890 92 9246 208 29 149 

Whiporie ·. .. P. elliotti ·. 1163 639 2367 1 718 152 5263 80 66 317 

Olney .. P. elliotti ·. 1041 649 2742 1120 422 6889 83 44 104 

Barcoongere .. .. P. taeda .. ·. 890 861 1 365 982 343 5112 70 67 362

TABLE 3 

Foliar phosphorus results from the three ashing methods 

Phosphorus (ppm) 

w 

o 

Forest Species Method A Method B Method C 

, 

Replicates ) Mean Replicates I Mean Replicates IMean 

I 

Mullions Range .. .. P. radiata .. 796 777 816 796 732 777 750 753 768 787 758 771 

Old Depot .. " P. radiata .. 2010 2001 2007 2006 1949 1926 1971 1949 2008 2033 1956 1999 

Penrose .. .. .. P. radiata .. 978 999 958 978 960 986 971 972 991 1001 989 994 

Jervis Bay .. .. P. radiata .. 1 891 1832 1679 1801 1863 1748 1746 1786 1863 1826 1837 1842 

Murraguldrie .. .. P. radiata .. 3394 3400 3436 3410 3307 3417 3258 3327 3431 3367 3368 3389 

Belanglo .. .. .. P. radiata .. 1081 998 1110 1063 1063 1005 1087 1052 1079 1069 1019 1056 

Mannus .. .. .. P. radiata .. 2574 2513 2655 2581 2504 2561 2440 2502 2531 2568 2566 2555 

Lidsdale .. .. .. P. radiata .. 1701 1660 1643 1668 1692 1 635 1 635 1673 1671 1659 1690 1673 

Newnes .. .. P. radiata .. 1240 1220 '1177 1212 1144 1152 1131 1142 1203 1168 1156 1176 

Whiporie :: .. .. P. e//iottii .. 1132 1171 /1185 1163 1179 1126 1119 1141 1169 1 159 1139 1156 

Olney .. .. P. e//iottii .. 1067 1 018 1 038 1041 1029 1115 1016 1053 1064 1073 1090 1076 

Barcoongere .. .. P. taeda .. .. 879 879 912 890 878 887 862 876 846 880 881 869 

-- ------ 

Grand Mean .. .. .. 1551 1 519 1546

ABLE 3A 

Analysis ofv.7riance for foliar phosphorus results from the three aslzing methods 

Dispersion 

lDegrees of freedomI Sum of squares 

I 

Variances 

F ratio 

~.... 

Between ashing methods .. .. .. . , .. .. 2 15660'4 7830·19 

Between forests .. .. .. .. .. ·. 11 61849000 5622640 492,0** 

Interaction of methods .. .. .. .. .. ·. 22 284097 12913'5 .. 

Inter-method reproducibility (error) .. .. .. ·. 72 822877-4 11428'9 .. 

Total .. .. .. .. .. .. ·. 107 62971 700 .. .. 

I i 

** Significant at 1 per cent level.

TABLE 4 

Foliar calcium results from the three ashing methods 

Calcium (ppm) 


Replicates Mean Replicates Mean Replicates Mean 

Mullions Range ... .. P. radiata ·. 895 992 948 945 854 907 808 856 958 951 908 939 

Old Depot ·. " P. radiata ·. 2176 2061 2254 2164 2249 2215 2169 2211 1964 2134 2128 207~ 

Penrose ·. ·. .. P. radiata ·. 4003 4038 4058 4033 3756 3824 3772 3784 4065 4024 3993 4027 

Jervis Bay ·. .. P. radiata .. 1 185 1156 1231 1191 1113 1175 1180 1156 1155 1189 1175 1173 

Murraguldrie I ., .. P. radiata ·. 3312 3083 3306 3234 3148 3255 3267 3223 3263 3315 3017 3198 

Belanglo ·. ·. " P. radiata ·. 1786 1714 1960 1820 1843 1 885 1831 1 853 1849 1769 1999 1872 

Mannus ·. ·. .. P. radiata ·. 2938 2921 2922 2927 3021 3066 3005 3031 2959 3000 3087 3015 

Lidsdale ·. ·. .. P. radiata ·. 2221 2418 2413 2351 2485 2366 2323 2391 2338 2343 2405 2362 

Newnes ·. .. P. radiata ·. 842 747 798 796 821 727 722 757 900 918 842 887 

Whiporie ., ·. .. P. e//iottii ·. 2348 2394 2360 2367 2221 2231 2608 2387 2527 1914 2516 2319 

Olney ·. .. P. e//iottii ·. 2751 2707 2767 2742 2756 2673 2635 2688 2576 2709 2833 2706 

Barcoongere ·. .. P. taeda.. · . 1315 1 329 1452 1 365 1407 1 288 1 333 1 343 1254 1403 1381 1346 

Grand Mean .. .. ·. 2161 2140 2160 

~ 

N

TABLE 4A 

Analysis of variance for foUar calcium results from the three ashing methods 

w 

c..> 

Dispersion Degrees of freedom Sum of squares Variances F ratio 

. 

Between ashing methods .. .. .. .. .. .. 2 171 388 85694'1 1·41 

Between ,forests .. .. .. .. .. .. 11 94016327 8546940 140'9** 

Interaction of methods .. .. .. .. .. .. 22 1 123900 51 086'5 .. 

Inter-method reproducibility (error) .. .. .. .. 72 2851120 60663-9 .- 

Total .. .. .. .. .. .. .. 107 99679420 .- .. 

** Significant at 1 per cent level. 

1 ,

TABLE 

Foliar potassium results from the three ashing procedures 

Potassium (ppm) . 


Replicates IMean Replicates I Mean Replicates I Mean 

~ 

Mullions Range 

" 

.• P. radiata .. 5749 6363 6932 6348 5613 6066 6148 5942 6425 6363 6377 6388 

Old Depot 

" 

.. P. radiata .. 5646 5360 5442 5483 5460 5266 5322 5349 5545 5232 5296 5358 

Penrose .. .. .• P. radiata .. 5557 6179 6270 6002 6174 6031 5919 6043 6182 5903 6110 6065 

Jervis Bay .. .. P. radiata .. 8121 9055 9163 8780 8967 9342 8903 9071 8175 8930 8837 8647 

Murraguldrie I .. ., P. radiata .. 5222 5691 5651 5521 5391 5085 5379 5285 5483 5572 5175 5410 

Belanglo .. .. .• P. radiata .. 6834 7094 6210 6713 7072 6585 6672 6776 7043 6354 6615 6671 

Mannus .. .. •. P. radiata .. 5730 6069 6133 5977 6059 5983 5903 5982 6085 5874 5870 5943 

Lidsdale .. 

" 

.. P. radiata .. 8932 8897 8929 8919 8956 8854 8810 8873 8342 8801 9071 8738 

Newnes 

" 

.• P. radiata .. 9396 9338 9004 9246 9392 7889 8473 8585 7389 7963 6881 7411 

Whiporie :: .. •• P. elliottii .. 5539 4872 5377 5263 4797 5196 6181 5391 5274 5231 5131 5212 

Onley 

" 

.. P. elliottii .. 6836 6857 6974 6889 7424 7556 5928 6969 6913 7130 7377 7140 

Barcoongere 

" 

.. P. taeda.. .. 4425 5311 5600 5112 5311 4713 5156 5060 4735 5590 5422 5249 

-- -------------------~-------------- 

Grand Mean .. .. .. 6688 6611 6519

TABLE 5A 

Analysis ofvariance for foliar potassium results from the three ashing methods 

Dispersion IDegrees of freedomI Sum of squares I Variances F ratio 

w Between ashing methods .. 

VI 

.. .. .. ·. .. 2 511 591 255796 1-81 

Between forests .. .. .. .. ·. " 

11 189577 903 17234355 122'13** 

Interaction of methods .. .. .. .. ·. .. 22 5 no 541 260025 1'84 

Inter-method reproducibility (error) .. .. .. .. n 10 160 174 141114 .. 

--------- 

Total .. ., .. .. .. .. .. 107 205970209 .. .. 

** Significant at 1 per cent level.

TABLE 6 

Foliar aluminium results from the three ashing methods 

Aluminium (ppm) 


Replicates IMean Replicates IMean Replicates I Mean 

l.J) 

Cl\ 


Old Depot .. .. P. radiata .. 575 541 551 556 428 404 410 414 688 633 612 644 



Murraguldrie I .. .. P. radiata .. 716 746 738 733 814 854 813 827 741 748 783 757 



Lidsdale .. .. .. P. radiata .. 925 1057 843 942 918 957 881 919 995 1010 959 988 

Newnes .. .. .. P. radiata .. 717 762 759 746 817 699 684 733 655 808 745 736 

Whiporie .. .. .. P. elliottii .. 646 648 624 639 517 510 540 522 889 631 681 734 

Olney .. .. P. elliottii .. 647 671 628 649 610 627 636 624 639 650 643 644 


-- --------------------------------- 

Granj Mean .. .. .. 739 708 776 

_-

TABLE 7 

Foliar magnesium results from the three ashing methods 

Magnesium (ppm) 


Replicates I Mean Replicates I Mean Replicates I Mean 

w 

00 

Mullions Range ·. .. P. radiata .. 1078 1066 1082 1075 973 966 985 975 1079 998 1154 1077 

Old Depot ·. .. P. radiata .. 1094 1200 1027 1107 1089 1075 1047 1070 1232 1130 964 1109 


Jervis Bay .. .. P. radiata .. 957 985 98+ 955 877 911 914 901 967 848 934 916 

Murraguldrie I ·. .. P. radiata .. 2952 3120 3156 3076 2907 3017 2628 2851 2968 3002 3058 3009 




Newnes .. .. P. radiata .. 938 905 947 890 907 1004 1001 971 1036 1056 959 1017 

Whiporie .. .. .. P. elliottU .. 1732 1714 1709 1 718 1588 1522 1754 1621 1719 1767 1781 1756 


Barcoongere .. ., P. taeda .. .. 943 942 1060 982 924 919 870 904 1002 960 977 980 

--------------------------------- 

Grand Mean .. .. .. 1657 1568 1644 

1 _

TABLE 7A 

Analysis of variance for foliar magnesium results from the three ashing methods 

Dispersion IDegrees of freedomI Sum of squares I Variances F ratio 

w Between ashing metho:ls ., .. 

\0 Between forests . . . . . . 

Interaction of methods . . . . 

Inter-method reproducibility (error) 

2 

11 

22 

72 

174491 

58566767 

188533 

425492 

87245·5 

5324250 

8569·7 

5909·6 

14'76** 

900'95** 

1·45 

Total 

107 

59355300 


Significant difference required between ashing method means = 36·2.

TABLE 8 

Foliar sodium results from the three ashing methods 

Sodium (ppm) 


Replicates Mean Replicates Mean Replicates Mean 

.r>. 

o 


Old Depot ·. .. P. radiata .. 676 673 639 662 558 569 573 566 580 584 593 586 

Penrose .. ·. .. P. radiata .. 152 159 159 157 151 144 135 143 137 141 143 140 

Jervis Bay .. .. P. radiata .. 1960 1900 1940 1933 1620 I 761 1700 1694 1740 1600 1600 1647 

MurraguJdrie I .. .. P. radiata .. 18 39 31 29 33 28 34 32 34 38 51 41 


Mannus .. ·. .. P. radiata .. 66 59 49 58 29 29 33 30 36 35 34 35 

Lidsdale .. ·. .. P. radiata .. 186 185 171 181 177 175 174 175 185 211 251 216 

Newnes .. .. P. radiata .. 82 97 97 92 97 97 106 102 112 102 98 104 

Whiporie .. .. .. P. elliotti .. 151 161 145 152 134 143 142 140 125 120 134 126 

Olney .. .. P. elliotti .. 425 431 410 422 358 370 375 368 356 384 371 370 


Grand Mean .. .. .. 357 313 316 

",--------

TABLE 8A 

Analysis ofvariance for foliar sodium results from the three ashing methods 


.I>. 

Between ashing methods ·. .. .. · . · . ·. 2 43607 21804 47,19** 

Between forests · . .. ·. ·. · . ·. I1 22772 614 2070238 4481,03** 

Interaction of methods .. .. ·. · . · . · . 22 130851 5948 12'87** 

Inter-method reproducibility (error) ·. ·. ·. ·. 72 33255 462 .. 

----------------------------------- 

Total . . · . .. ·. · . · . · . 107 22980327 .. ., 

** Significant at I per cent level. 

Significant difference required between ashing method means = 10'I.

TABLE 9 

Foliar manganese results from the three ashing methods 

Manganese (ppm) 


! 

- 

Replicates I Mean Replicates IMean Replicates I Mean 

~ 

Mullions Range .. •. P. radiata · , 101 101 101 101 102 109 109 '107 107 106 109 107 

Old Depot ·. ., P, radiata , . 203 208 207 206 207 209 206 207 208 211 213 211 

Penrose ·. .. .. P. radiata ·. 283 277 291 284 281 294 288 288 294 299 305 299 

Jervis Bay ·. .. P. radiata ·. 35 36 37 36 31 33 36 33 35 37 34 35 

Murraguldrie I .. .. P. radiata ·. 313 323 316 317 315 319 318 317 334 334 335 334 

Belanglo · , ·. .. P. radiata .. 142 137 139 139 141 145 145 142 139 141 139 140 

Mannus ·. .. .. P. radiata .. 456 458 429 448 443 428 448 440 451 458 447 452 

Lidsdale ·. ·. .. P, radiata · , 376 374 379 376 367 345 357 356 391 401 398 397 

Newnes ·. .. P. radiata .. 154 145 148 149 142 145 152 146 175 174 160 170 

Whiporie ,. .. ., P. elliottii ·. 318 321 313 317 303 210 162 225 337 332 340 336 

OIney ·. .. P. elliottii ·. 103 103 106 104 96 55 47 66 106 113 115 1P 

Barcoongere ·. .. P. taeda .. ·. 363 352 372 362 352 354 351 352 349 350 353 351 

------------------------------- 

Grand Mean .. 

" ·. 237 223 245

TABLE 9A 

Analysis ofvariance for foliar manganese results from the three ashing methods 

Dispersion IDegrees of freedomI Sum of squares 

I 

Variances 

F ratio 

ol> 

~ 

Between ashing methods .. .. ·. .. ., ·. 2 8798 4399 23,03** 

Between forests .. .. ·. .. ·. ·. 11 1676789 152435 798,09** 

Interaction of methods .. .. ·. ·. 22 21034 956 5·01** 

Inter-method reproducibility '(error)' ·. ... ·. ·. 72 13 759 191 .. 

Total .. .. ., .. .. ·. ·. 107 1720380 .. .. 


Significant difference required between ashing method means = 7.

TABLE 10 

FoliaI' iron results from the three aslzing methods 

\ 

Iron (ppm) 


t 

Replicates Mean Replicates 

I 

Mean Replicates Mean 

Mullions Range ·. P. radiata ·. 252 257 255 255 251 268 265 261 278 281 282 280 

Old Depot ·. .. P.radiata · . 239 233 231 . 234 233 234 231 233 252 248 258 253 

Penrose · . ·. .. P. radiata .. 197 193 206 199 208 209 204 207 225 232 204 220 

Jervis Bay ·. P. radiata · . 354 352 325 344 366 327 325 339 325 328 331 328 

Murraguldrie I ·. .. P. radiata ·. 185 185 J80 183 191 185 189 188 18J 196 192 190 

Belanglo · . ·. .. P.radiata ·. 200 225 207 211 222 253 200 225 225 224 218 222 

Mannus · . ·. .. P.radiata ·. J72 164 170 J69 159 160 158 J59 174 172 158 168 

Lidsdale · . ·. .. P. radiata ·. 153 J55 139 149 148 143 150 J47 140 176 168 161 

Newnes ·. ·. .. P.radiata · . 211 205 207 208 201 173 206 J93 190 187 171 183 

Whiporie · . ·. .. P. elliottii .. 72 83 86 80 87 83 65 78 90 85 92 89 

Olney .. ·. .. P. elliottii ·. 91 79 80 83 101 59 71 77 80 100 109 96 

Barcoongere ·. .. P. taeda .. ·. 64 75 70 70 84 77 73 78 77 71 81 76 

Grand Mean .. .. 

" ·182 182 189

TABU lOA 

Analysis ofvariance for foUar iroll results from the three ashing methods' 


.j>.. 

V\ 

Between ashing methods . , .. .. .. .. .. 2 1117 559 4'82** 

Between forests .. .. .. .. .. .. 11 615839 55985 482,63** 

Interaction of methods .. .. .. .. .. .. 22 4584 208 1·79 

Inter-method reproducibility (error) .. .. .. .. n 8339 116 .. 

Total .. .. .. .. .. .. .. 107 62987) .. .. 

** Significant at 1 p~r cent level. 

Significant difference required between asking method means = 5·1.

TABLE 11 

Foliarlzinc results from the three ashing methods 

Zinc (ppm) 


Replicates IMean Replicates IMean Replicates I Mean 

~ 

0'\ 


Old Depot .. .. P. radiata .. 81 78 78 79 79 77 75 77 76 77 75 76 



Murraguldrie I .. .. P. radiata .. 101 107 98 102 99 98 102 100 87 89 90 89 




Newnes .. .. .. P. radiata .. 30 28 29 29 29 29 29 29 31 28 26 28 

Whiporie .. .. .. P. elliottii .. 67 66 65 66 70 69 68 69 67 64 66 66 


Barcoongere .. .. P. taeda.. .. 67 67 66 67 65 62 62 62 67 65 68 67 

-. -------------------------------'-- 

Grand Mean .. .. .. 65 65 62

TABLE 11A 

Analysis ofvariance for foliar zinc results from the three ashing methods 

~ 

Dispersion 

IDegrees of freedomI Sum of squares 

Variances 

F ratio 

- 

7** 

4** 

** 

Between ashing methods .. .. .. .. .. .. 2 162 81 11'5 

Between forests .. .. .. .. .. 11 54524 4957 708·1 

Interaction of methods .. .. .. .. .. .. 22 571 26 3'1 

Inter-method reproducibility (error) .. .. ., .. n 491 7 .. 

Total .. .. .. .. .. .. .. 107 55748 .. .. 


Significant difference required between ashing method means = 1·2.

TABLE 12 

Foliar chemical analyses obtainedfrom ash solutions (offoliage samples from Nalbaugh S.F.) prepared according to ashing methods A and C 

~ 

00 

Ashing procedure 

I 

Sample 

Plot N,o., 

P 

I 

AI 

I 

Ca 

I 

Mg 

ppm 

I 

Na 

I 

K 

·1 

Fe 

I 

Mn 

I Zn 

Method C .. .. .. 3 3079 509 1937 2792 387 8857 133 161 98 

5 3057 533 1921 2085 443 8583 135 113 85 

7 2960 515 2130 2576 501 6414 190 121 80 

12 1646 612 2056 2731 407 7419 160 119 77 

14 2497 811 1157 1646 550 11 804 167 158 77 

15 1576 680 3479 4434 425 4342 141 229 131 

18 1782. 457 872 1161 639 5567 123 137 48 

22 1445 444 1395 1674 767 6027 143 3]8 69 

------------------------------- 

Mean 2255 570 1 868 2387 514 7376 149 169 83 

--------------------------------------- 

Method A .. .. .. 3 3020 513 . 2035 3070 397 9497 128 170 105 

5 2868 443 1 733 1 939 327 7961 142 108 73 

7 3004 642 2188 2670 541 6788 214 125 96 

12 1671 558 1969 2599 342 7462 148 120 82 

14 2460 762 1020 1607 525 12244 135 177 81 

15 1669 630 3388 4230 439 4451 127 225 94 

18 1792 337 1036 1 315 556 6451 134 168 55 

22 1469 449 1524 1730 678 6517 133 299 75 

---- 

Mean 2244 541 1861 2395 476 7671 145 174 83

TABLE 13 

Mean chemical analyses obtained from ash solutions prepared by method A atvarious 

ashing temperatures 

ppm 

Ashing 

temperature 

Cc) P Al Ca Na K Fe Zn Mn 

I I I Mg I , 

I I I 

350 ·. ·. 800 674 2926 1420 192 3860 52 27 142 

400 ·. ·. 794 670 2987 1436 189 3174 50 27 146 

425 .. ·. 786 720 2968 1422 187 3867 51 26 147 

450 ·. ·. 786 721 2994 1403 189 3587 52 26 147 

475 ., ·. 786 722 2987 1387 187 3578 50 26 148 

500 ·. ·. 787 722 2977 1385 189 3519 60 25 149 

525 .. ·. 802 719 2997 1356 189 3618 57 26 148 

550 ·. ·. 801 678 2948 1368 187 3610 55 27 145 

------------------ 

LSD 0·05 .. .. .. 40 .. .. 131 .. .. .. 

TABLE 14 

Phosphorus results obtainedfrom ash solutions prepared by methodA at various ashing-- 

temperatures 

Foliar phosphorus (ppm) 

Ashing 

temperature 

CC) Replicates Mean 

350 •. .. .. .. 813 786 802 800 

400 •• .. .. .. 814 776 792 794 

425 .. .. .. .. 785 801 772 786 

450 .. .. .. .. 800 781 777 786 

475 .. .. .. .. 782 776 800 786 

500 .. .. .. .. 793 772 796 787 

525 .. .. .. .. 794 811 801 802 

550 .. .. .. .. 802 814 787 801 

I 

No significant differences between ashing temperatures. 

49

TABLE 15 

Foliar aluminium results obtained from ash solutions prepared by method A at various 


Foliar aluminium (ppm) 

Ashing 

temperature 

Cc) Replicates Mean 

350 .. .. .. .. 661 650 711 674 

400 .. · . · . · . 692 670 649 670 

425 " ·. · . · .. 734 715 711 720 

450 .. · . ·. · . 714 732 716 721 

475 .. .. .. · . 755 717 694 722 

500 .. · . .. ·. 733 720 714 722 

525 .. ., .. ·. 757 661 739 719 

550 .. .. .. .. 712 646 676 678 

/ 


TABLE 16 

Foliar calcium results obtained from ash solutions prepared by method A at various 


Foliar calcium (ppm) 

Ashing 

temperature 

Cc) Replicates Mean 

350 .. .. .. .. 2948 2912 2918 2926 

400 .. .. .. .. 2992 2952 3017 2987 

425 " .. .. .. 2957 2991 2956 2968 

450 " .. .. .. 2987 2996 2999 2994 

475 .. .. .. .. 3002 2997 2962 2987 

500 .. .. .. .. 2985 2991 2955 2977 

525 " .. .. .. 3012 2998 2981 2997 

550 .. .. .. .. 2948 2912 2984 2948 

I 

LSD 0.0;; = 40. 

50

TABLE 17 

Foliar magnesium results from ash solutions prepared by method A at various ashing 

temperatures 

Foliar magnesium (ppm) 

Ashing 

temperature 

eC) Replicates Mean 

350 .. .. .. .. 1438 1392 1430 1420 

400 .. .. .. .. 1382 1442 1484 1436 

425 .. .. .. .. 1387 1449 1430 1422 

450 .. .. .. .. 1436 1384 1389 1403 

475 .. ... .. .. 1368 1426 1367 1387 

500 .. .. .. .. 1372 1 363 1420 1385 

525 .. .. .. .. 1428 1335 1305 1356 

550 .. .. .. .. 1352 1410 1342 1368 

I 


TABLE 18 

Foliar sodium results from ash solutions prepared by method A at various ashing 

temperatures 

Foliar sodium (ppm) 

Ashing 

temperature 


350 .. .. .. .. 203 183 190 192 

400 .. .. .. .. 191 183 193 189 

425 .. .. .. .. 195 183 183 187 

450 .. .. .. .. 188 189 191 189 

475 .. .. .. .. 186 188 188 187 

500 .. .. .. .. 196 186 185 189 

5,25 .. .. .. .. 189 188 189 189 

550 .. .. .. .. 184 191 186 187 

I 


51,

TABLE 19 

Foliar potassium results obtained from ash solutions prepared by method A at various 


Ashing 

temperature 

Cc) 

Foliar potassium (ppm) 

Replicates 

Mean 

350 .. 

400 .. 

425 .. 

450 .. 

475 .. 

500 .. 

525 .. 

550 .. 

3910 

3878 

3967 

3678 

3598 

3536 

3637 

3662 

3802 

3710 

3831 

3510 

3475 

3573 

3560 

3628 

3868 

3734 

3801 

3573 

3661 

3448 

3658 

3539 

3860 

3774 

3867 

3587 

3578 

3519 

3618 

3610 

LSD 0.0;; = 131. 

TABLE 20 

Foliar iron results obtained from ask solutions prepared by method A at various asking 

temperatures 

Foliar iron (ppm) 

Ashing 

temperature 

(0C) Replicates Mean 

350 .. 54 52 50 52 

400 .. 50 50 51 50 

425 .. 53 50 50 51 

450 .. 55 51 50 52 

475 .. 54 49 47 50 

500 .. 68 52 59 60 

525 .. 66 50 55 57 

550 .. 62 51 52 55 


52

---------------------------------------- 

TABLE 21 

.Foliar zinc results obtainedfrom ash solutions prepared by method A at various ashing 

temperatures 

Foliar zinc (ppm) 

Ashing 

temperature 


350 .. .. .. .. 30 28 23 27 

400 .. .. .. .. 25 28 28 27 

425 .. .. .. .. 26 27 26 26 

450 .. .. .. .. 27 26 26 26 

475 .. .. 

" 

.. 27 26 26 26 

500 .. .. 

" 

.. 25 24 25 25 

525 .. .. .. .. 27 24 27 26 

550 .• .. .. .. 26 27 27 27 


TABLE 22 

.Foliar manganese results obtained from ash solutions prepared by method A at various 

temperatures 

Foliar manganese (ppm) 

Ashing 

temperature 

(0C) Replicates Mean 

350 .. .. .. .. 143 134 149 142 

400 .. .. .. .. 142 144 152 1.16 

425 .. .. .. .. 144 147 149 147 

450 .. .. .. .. 148 147 146 147 

475 .. .. .. .. 149 151 144 148 

500 .. .. .. .. 152 147 149 149 

525 .. .. .. .. 157 141 147 148 

550 .. .. .. .. 146 146 144 145 


53

TABLE 23 

Mean foliar analysis obtained using method A with various ash-dissolving solutions 

Ash-dissolving 

soultions 

ppm 

P Al Ca 

I I I Mg I 

Na K Fe Zn Mn 

I I I I 

1:1 HCl .. / 801 67612948 1368 187 3610 55 26·7 145 

1:1 HCI-three 

digestions . '1 778 700 2917 1377 188 3577 58 24'0 146 

1:1 HNOs .. 801 688 2955 1372 188 3516 56 25·0 143 

1:1 HCI-HNOs 

(50% v/v) .. 1 787 673 2941 1 337 189 3639 54 26·0 146 

LSD 0.05 "1-"---"-.. 

l-··-.. -.. -.. ~-.. 

TABLE 24 

Foliar phosphorus results obtained using method A with various ash-dissolving 

solutions 

Ash-dissolving solutions 

1 1 HCl .. .. .. .. 802 

/ 

Phosphorus (ppm) 

Replicates 

l Mean 

, 

787 801 

1 1 HCI-three digestions .. .. 773 l814 

~93 

769 7"78 

1 1 HNOs .. .. .. .. 808 , ~4 

/ 811 801 

1 1 HCl-HNOs (50% v/v) .. 799, 783" 778 787 

. / 

No significant difference between treatments. \. 

54 

/

TABLE 25 

Foliar aluminium results obtained using method A with various ash-dissolving solutions 


Aluminium (ppm) 

Replicates 

I Mean 

1:1 HCl 

" " 

.. 

" 

712 646 676 676 

1:1 HC1-three digestions,. 

" 

709 723 667 700 

1:1 HNOs ., .. 

" " 

721 688 655 688 

1:1 HCI-HNOs (50 per cent v/v) .. 705 643 672 673 

No significant differences between treatments, 

TABLE 26 

Foliar calcium results obtained using method A with various ash-dissolving solutions 


1:1 HCl .. .. .. .. 

1:1 HC1.....:.three digestions ,", , . 

1:1 HNOs.. ., ,. .. 

1:1 HCI-HNOs (50 per cent v/v)., 

Calcium (ppm) 

Replicates 

I Mean 

2948 2912 2984 2948 

2920 2944 2887 2917 

2948 2980 2937 2955 

2910 2978 2935 2941 

No significant differences between treatments. 

55

- -------------------- 

TABLE 27 

Foliar magnesium results obtained using method A with various ash-dissolving 

solutions 


Magnesium (ppm) 

Replicates 

Mean 

1:1 HCl .. .. .. .. 1352 1410 1342 1368 

1:1 HCl-three digestions .. o. 1368 1367 1396 1377 

1:1 HNOa o. .. .. .. 1362 1390 1364 1372 

1:1 HCI-HNOa .. .. .. 1371 1335 1305 1337 


TABLE 28 

Foliar sodium results obtained ([sing method A with various ash-dissolving solutions 

Ash-dissolving $olutions 

Sodium (ppm) 

Replicates 

Mean 

1 1 HCl .0 •• •• •• 

1 1 HCl-three digestion 00 o' 

1 1 HNOa .. .. ., .. 

1 1 HCI-HN03 (50 per cent v/v) .. 

184 l Q l 186 187 

186 186 191 188 

188 189 188 188 

190 188 190 189 


56

TABLE 29 

Foliar potassium results obtained using method A with various ash-dissolving solutions 


Potassium (ppm) 

Replicates 

I Mean 

1:1HC1 .. .. .. .. 

1:1 HC1-three digestions.. .. 

'1:1 HNOs .. .. .. .. 

il:1 HC1-HNOs (50 per cent v/v) .. 

3662 

3743 

3527 

3495 

3628 

3523 

3556 

3745 

3539 

3466 

3466 

3676 

3610 

3577 

3516 

3639 


TABLE 30 

Foliar iron results obtained using method A with various ash-dissolving solutions 


Replicates 

Iron (ppm) 

Mean 

1:1 HC1 .. .. .. .. 62 51 52 55 

1:1 HC1-three digestions .. .. 65 56 52 58 

1:1 HNOs .. .. .. .. 65 52 52 56 

1:1 HCI-HNOs (50 per· cent v/v) .. 51 53 58 54 

No sigpificant differences between treatments. 

57

TABLE 31 

Foliar zinc results obtained using method A with various ash-dissolving solutions 

Ash-dissolving solution 

Replicates 

Zinc (ppm) 

I Mean 

1:1 HCl ·. ·. ·. .. 26 27 27 26·7 

1:1 HCl-three digestions .. ·. 25 24 23 24'0 

1:1 HNOs ·. · . ·. ·. 25 25 25 25·0 

1:1 HCI-HNOs (50 per cent v/v) .. 27 25 26 26·0 

LSD 0.05 = 1,4. 

TABLE 32 

Foliar manganese results obtained using method A with various ash-dissolving solutions 


Manganese (ppm) 

Replicates 

I Mean 

1:1 HCl · . ·. ·. ·. 146 146 144 145 

1:1 HCl-three digestions .. ·. 148 144 145 146 

1:1 HNOs ·. ·. ·. ·. 140 145 143 143 

1:1 HCI-HNOs (50 per cent v/v) .. 148 144 145 146 


58

Results from recovery experiments. 

TABLE 33 

In the case ofthe foliage only results-these are actual determinations. 

based on the non-muffled samples 

The other results are precentage recoveries 

Ashing 

Temp- 

Number Sample erature 

(OC) 

P Al Ca Mg Na K Fe Zn Mn· 

Ut 

\0 

3 Elements .. .. 420 99'9 100'4 96'2 99'8 97'2 100·4 99·4 100·2 98'6 

4 Elements + Si .. ., 420 97·0 100'8 93'0 96'6 113'1 101'5 99'1 101'5 97'8 

5 Foliage (p.p.m.) .. ., 420 975 1086 1943 1344 498 3556 153 39 387 

6 Foliage + Elements .. 420 99·9 100·1 102'9 100'5 98-1 103·2 72'1 101'5 99'1 

7 Foliage + Elements + Si .. 420 96·6 102·8 102'4 101'2 79'9 101·3 63·2 100·0 95'3 

8 Elements .. .. 500 101·3 100'8 94'3 101·0 95'7 98·5 106·0 101·5 99·7 

9 Elements + Si .. .. 500 99·4 98'8 92-3 94'4 103-3 98'4 99·1 94·0 90'5 

10 Foliage (p.p.m.) . . . . 500 974 1102 1950 1354 504 3464 157 38 385 

11 Foliage + Elements .. 500 101·7 99·7 105'2 99·8 95·0 102·2 93·0 95-3 100·0 

12 Foliage + Elements + Si .. 500 100·9 102'0 98'4 100'9 83'7 102·0 89'3 76'4 92·4 

13 Elements ., .. 550 100·0 98·8 93'0 98·5 85'2 105·7 97'9 100·6 91·S 

14 Elements + Si ., .. 550 100·6 97'3 93-8 94·5 101·6 92'4 99·1 99·2 92'4 

15 Foliage (p.p.m.) .. .. 550 958 1048 1915 1360 473 3557 125 38 367 

16 Foliage + Elements .. 550 100·1 103·8 99'1 100·2 64·8 100·2 87·6 88·2 93·3 

17 Foliage + Elements + Si .. 550 97'6 98·5 97·7 95'0 80·6 98'5 51'5 79·6 87·0 

- 

18 Elements .. .. 600 100'6 99·0 96'5 100'3 95'2 101·1 107·2 105'3 99·1 

19 Elements + Si .. .. 600 96·6 101·5 91'3 94·8 93'2 84·0 99·1 84·7 97'2 

20 Foliage (p.p.m.) .. .. 600 972 1033 1904 1353 465 3257 140 35 388 

21 Foliage + Elements .. 600 102'1 106·3 98'1 100·4 87'5 92·5 96'6 86·9 94·1 

22 Foliage + Elements + Si " 600 100'7 101·4 96'7 97'5 93·6 90·6 67·8 69·7 72·4

APPENDIX 1. Determination of phosphorus in foliage material. 

REAGENTS 

Reagent A-76.8g ,ammonium molybdate (A.R.) is dissolved in approx. 

200ml distilled water. 1.7555g antimony potassium tartrate is dissolved 

in approx. 100ml distilled water. 896m1 36N H 2S04 is dissolved 

in 500ml distilled water. The first two reagents are added to the dilute 

sulphuric acid and the solution diluted to 2 litres with distilled water. 

This solution is stored in a refrigerator. 

Reagent B-1.70g ascorbic acid is dissolved in 100ml distilled water, 

50ml reagent A are added and diluted to 200ml with distilled water. 

This reagent must be prepared fresh daily. 

2N h~S04-Approximate1y 55ml 36N H 2S04 are added to 500ml 

distilled water and diluted to 1 litre with distilled water. 

2N NaOH-80g NaOH are dissolved in distilled water and diluted to 

1 litre. 

2, 6-dinitrophenol indicator-0.25g indicator is dissolved in a few 

drops of alcohol (lml) and diluted to 100ml with distilled water. 

Standard phosphorus solution-(50 ppm and 5 ppm) 0.2195g 

KH 2 P04 is dissolved in approximately 100ml distilled water, 5ml 36N 

H 2S04 added and the solution diluted to 1 litre with distilled water. 

This stock solution contains 50 ppm P. SOml of this solution are 

diluted to 500ml to give a working standard of 5 ppm P. 

PROCEDURE 

An aliquot is taken from each of the ash solutions obtained by the 

ashing methods. It is preferable for the aliquot to contain 30-50 p.g P 

and hence a 2ml or Sml aliquot is the most suitable. The aliquot is 

placed in a SOml volumetric flask and distilled water added to

--~~~------------------------ ---------- 

STANDARD CuRVE 

Aliquots containing 10, 20, 30, 40, 50 P,g P are placed in separate: 

50ml volumetric flasks. If an automatic dilutor has been used for the 

samples, then the same procedure should be followed for the standards, 

by taking 2ml aliquots by the sample syringe from solutions containing 

5, 10, 15, 20, 25, 30 ppm P. The procedure for colour developmentis 

then followed as above. 

It is not necessary to determine all the points on the standard 

curve with each set of foliar ash solutions. Only one or two points on' 

the graph need be checked with each set of estimations. A new curve 

~s determined when a new batch of reagent A is prepared. 

The absorbances obtained are plotted against the p,g P in the all.... 

quots. A straight line is drawn through the points and should pass. 

through the origin. The equation of the lirte is determined for ease of 

calculation. 

CALCULATION 

ppm. P = 

(/log Pin aliquot) x Ash Volume 

Aliquot x a.D. wt foliage 

61

APPENDIX 2. Determination of aluminium in foliage material. 

REAGENTS 

10 per cent Ammonium Acetate-100g ammonium acetate (A.R.) is 

dissolved in 1 litre distilled water and the pH of the solution is adjusted 

to 5.3. 

0.2 per cent Ferron-2g ferron (8-hydroxy-7-iodoquinoline-5-sulphonic 

acid) is dissolved -in 1 litre distilled water. 

Standard Aluminium Solution-0.175 9g aluminium potassium sulphate 

(A.R.-A1K(S04)2.12H20) which has been stored in a desiccator over 

dry silica gel for a few days, is made up to 1 litre with distilled water. 

This solution contains 10 ppm AI. 

Standard Iron Solution-0.864 Og ferric ammonium SUlphate 

(AR.-FeNH4(S04h12H 2 0) is dissolved in sufficient water, 10 ml10N 

HCI is added and diluted to 1 litre with distilled water. This stock 

solution contains 100 ppm Fe. 50ml of ,this solution are diluted to 

500ml to give a solution of 10 ppm Fe. 

PROCEDURE 

An aliquot (usually'5ml) of the ash solution containing no more 

than 50 P,g AI, is placed in a 25 ml volumetric flask. 5ml ammonium 

acetate is added, followed by 2ml ferron. The solution is made to 

volume with distilled water ~md the absorbances determined at 370 

mp, and 600 m,." after standing for 1 hour. The developed colour is 

stable for approximately 24 hours. The absorbances are read against 

a blank containing distilled water to which reagents have been added 

as above. 

If an automatic dilutor is available, the sample syringe is set to 

take a 5ml aliquot from the ash solution and this is delivered into a 4" 

specimen tube with 5ml ammonium acetate from the diluent syringe. 

In the next operation, 2ml ferron is taken up by the sample syringe 

and delivered into the tube with 13ml' distilled water. The solution is 

mixed and read as above. 

STANDARD CURVES 

Aliquots containing 10 to 60 p,g Al are placed in 25ml volumetric 

flasks and additional aliquots containing 10 to 60 p,g Fe are placed 

in separate flasks. 5ml 10 per cent ammonium acetate is added followed 

by 2ml ferron. The solutions are made to volume with distilled 

water and the absorbances read at 370 mp, and 600 m,." against a 

reagent blank. 

If the automatic dilutor has been used, 5ml aliquots are taken 

from the appropriate standard solution and the procedure followed as 

for ash solutions. 

The total p,g of Al or Fe in the 25ml volume is plotted' against the 

E. reading. Straight lines are drawn through the points and pass through 

the origin. The equation of each line is calculated, e.g., 

p,g Fe (370 mp,) = C x E. reading 

p,g Fe (600 mp,) D x E. reading 

p,g Al (370 mp,) = A x E. reading 

(N.B. The E. readings obtained for Al and for Fe at 370 mp, 

are perfectly additive.) 

CALCULATIONS 

D 

Comparable E. reading (B) = (E. reading at 600 mp,) x C 

for Fe at 370 mp, 

E. reading for Al only = (E. reading at 370 mp,) - CB) 370 mp,. 

P,g Al x Ash Volume 

pm Al 

- Aliquot x O.D. wt foliage 

62

APPENDIX 3. Determination of Ca, Mg, K, Na, Fe, Zn and Mu in 

foliage material. 

REAGENTS 

Strontium Chloride Solution-1O.1440 g strontium chloride (AR.) is 

dissolved in distilled water and diluted to 1 litre with distilled water. 

This solution contains 3333 ppm Sr. 

Standard Solutions 

1000 ppm. Stock Calcium Soilution-2.4975 g calcium carbonate AR. 

(dried at 550 0 

C for 4 hours) is dissolved in hydrochloric acid and 

diluted to 1 litre with distilled water. 

1000 ppm. Stock Magnesium Solution-1g magnesium ribbon (99.9 

per cent purity) is dissolved in hydrochloric acid and diluted to 1 litre 

with distilled water. 

1 000 ppm. Stock Sodium Solurtion-2.5419g sodium chloride AR. 

(dried at 110 0 

C for 4 hours) is dissolved in 1 litre distilled water 

containing 1ml ION HC1. 

1 000 ppm. Stock Potassium Solu1Jion-1.9069g potassium chloride 

AR. ·(dried at 500 0 

C for 4 hours) is dissolved in 1 litre distilled water 

containing 1ml10N HC1. 

1000 ppm. Stock Manganese Solution-2.8768g potassium permanganafe 

AR. (99.9 per cent purity) is dissolved in distilled water and 

decolourized with oxalic acid. The solution is then diluted to 1 litre 

with distilled water containing 1ml ION HC1. (The concentration is 

checked by standardization against arsenious oxide.) 

1 000 ppm. Stock Iron Solution-7.0225g ferrous ammonium sul· 

phate AR. (Fe(NH4h(S04h,6H20) is dissolved in 1 litre distilled 

water containing 1ml ION HCt. 

1.000 ppm. Stock Zinc Solution-1g zinc metal (99.9 per cent purity) 

is dissolved in hydrochloric acid and diluted to 1 litre with distilled 

water. 

(BDH, Merck or similar stock standard solutions (1 000 ppm) are 

also appropriate.) 

Working Standard Solutions 

Working standard solutions are prepared for calcium, magnesium and 

potassium to contain these three elements together in the one 'solution. 

These solutions are 130 prepared to also contain 3 000 ppm. Sr "(as 

SrCb). Working standard solutions are prepared for each of the other 

elements by taking suitable aliquots from the stock standard solutions, 

adding 1ml ION HC1 and diluting to 250ml with distilled water. 

PROCEDURE 

For the determination of calcium, magnesium and potassium, a 

5ml aliquot is taken from the a§h solution and diluted to 50ml with 

the strontium chloride solution. This gives a solution with a final concentration 

of 3000 ppm Sr. This solution is aspirated into the burner 

of the A.A 

Sodium, iron, zinc and manganese are determined directly on the 

ash solution. 

63

OPERATING CONDITIONS FOR VARIAN AA6, ATOMIC ABSORPTION 

SPECTROPHOTOMETER 

Lamp Slit Wavecurrent 

length Fuel 

(mA) ([L) 

(A) 

Calcium ·. ·. 5 100 4227 N 2 O/Acet. 

Magnesium ·. ·. 4 50 2852 Air/Acet. 

Sodium .. ·. ·. 5 50 5890 Air/Propane. 

Potassium ·. .. 10 300 7665 Air/Propane. 

Iron .. · . ·. 5 100 2483 Air/Acet. 

Zinc .. ·. ·. 10 300 2139 Air/Propane. 

Manganese ·. ·. 10 100 2975 Air/Acet. 

The burner height, fuel flow and lateral position of the burner are 

so adjusted to give maximum sensitivity for each element. 

CALCULATION 

ppm Element 

(in foliage) 

(ppm in soln) x Ash Volume x Dilution 

a.D. wt foliage 

LO. 181 D. West, Government Printer

National Library of Australia 

ISBN 072401041 6 

ISSN 0085·3984

Preparation of Plant Material for Estimating a Wide Range of Elements

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